Cationic polyphosphazene compound, polyphosphazenes-drug conjugate compound and method for preparing same

ABSTRACT

The present invention relates to a new class of cationic linear polyphosphazenes bearing as side groups a hydrophilic poly(ethylene glycol) and a spacer group selected from the group consisting of lysine, oligopeptides containing lysine, amino-ethanol, amino-propanol, amino-butanol, amino-pentanol and amino-hexanol, and the polyphosphazene-drug conjugates comprising hydrophobic anticancer drugs by covalent bonding and the preparation methods thereof. The present polyphosphazene-drug conjugates exhibit outstanding tumor selectivity and low toxicity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/125,543, filed Oct. 20, 2016, which is incorporated herein byreference in its entirety for all purposes.

TECHNICAL FIELD

The present invention relates to highly tumor selective andbiocompatible cationic linear polyphosphazene carrier polymers and theiranticancer drug conjugates, and a preparation method thereof.

BACKGROUND ART

Most of the anticancer drugs currently in clinical use for chemotherapyare monomeric compounds with a low molecular weight less than 1000 Da.Such monomeric low molecular weight anticancer drugs are well known tocause severe toxicities and side effects due to their non-selectivity totumor cells and tissue when injected intravenously, and furthermore,their short half-life less than a few hours during blood circulationlimits their sustainable efficacy. Therefore, the most critical keytechnologies to overcome in the new anticancer drug development is thetumor targeting technology for selective delivery of anticancer drugs tothe tumor site and timely releasing technology of the active componentof anticancer drugs in the tumor site. A great deal of efforts toovercome such limits have been made in the world for last decades, andas a result, it has been discovered that polymeric drug delivery systemsare one of the most efficient and practical ways to bring abreakthrough, from which a new field called “polymer therapy” emerged(R. Haag, F. Kratz, Angew. Chem. Int. Ed. 45 (2006) 1198-1215).

Most of the polymers employed as drug delivery systems are organicpolymers synthetic or natural. Numerous natural and synthetic polymerswere attempted as drug delivery systems for polymer therapy, but only alimited number of drug delivery systems were found to be useful, sincein addition to afore-mentioned tumor targeting properties and releasingkinetics many requirements such as water solubility, biodegradability,self-toxicity, compatibility with the loaded drug should be satisfiedfor polymer therapy of cancer.

The present inventors discovered decades ago that in contrast to theorganic polymers above-mentioned a new class of organic/inorganic hybridpolymers were designed by grafting various organic groups to theinorganic polymer backbone consisting of alternating nitrogen andphosphorus atoms called phospphazene (Y. S. Sohn, et al. Macromolecules,1995, 28, 7566), which have been intensively developed as drug deliverysystems for cancer therapy. In the early stage, various hydrophilicpoly(ethylene glycol) (PEG) and hydrophobic oligopeptides wereintroduced into the phosphazene backbone to obtain amphiphilicpolyphosphazenes affording thermosensitive drug delivery systems. It wasalso found that such amphiphilic polyphosphazenes are self-assembledinto various nanostructures such as thermosensitive micelles andhydrogels useful for drug delivery in aqueous solution, but also wereobserved decreased water solubility and some toxicity due to somehydrophobic oligopeptide grafted to polyphosphazene backbone. It isgenerally known that such amphiphilic polymers exhibit a lower criticalsolution temperature (LCST) at which the polymer start to precipitatefrom its aqueous solution when slowly heated. Therefore, amphiphilicpolymer drug delivery system should exhibit higher LCST than bodytemperature for intravenous injection to avoid its precipitation duringblood circulation.

Concerning the hydrophobic anticancer drugs, the taxane family includingpaclitaxel and docetaxel is one of the most widely used for efficientchemotherapy of a wide spectrum of cancers including breast, ovarian andnon-small cell lung cancers. Since these taxane anticancer agents areonly slightly soluble in water (<1 μg/ml), they cannot be directlyinjected but should be formulated using surfactants such as Polysorbate80 or Cremophore EL and ethanol for IV injection. However, suchformulated taxane anticancer agents exhibit several adverse effectsincluding neurotoxicity and neutropenia due to the agent itself andhypersensitivity due to the solvent system, which limit their widerclinical use.

Therefore, a great deal of researches have been made in various fieldsduring the last decade to overcome such adverse effects, and among them,nanotechnology using various structural morphology is most activelyprogressing. In particular, the polymeric micelles composed of thehydrophilic outer shell and hydrophobic core can afford to solubilizethe hydrophobic anticancer drugs such as taxane by encapsulation of thehydrophobic drug molecules in the hydrophobic micelle core. Also, thetaxane drug molecules may be conjugated by chemical bonding to thehydrophilic poly(ethylene glycol) to solubilize, which are now inclinical trials.

Such polymeric prodrugs composed of small molecular anticancer drugsconjugated to the polymeric drug delivery systems are expected to extendtheir blood circulation time and afford tumor targeting properties byenhanced permeability and retention (EPR) effect (H. Maeda et al. J.Control. Release 65(2000) 271-284) along with controlled drug release,resulting in maximum drug efficacy and minimum toxicity. According tothe recent reports, the polymer particle size should be in the range of50-200 nm in order to exhibit tumor targeting properties by EPR effect(V. P. Torchilin, J. Control. Release 73 (2001) 137-172). Also, it wasreported from the study of gene delivery that cationic polymers caneasily permeate anionic tumor cells (N. P. Gabrielson, D. W. Park, J.Control. Release 136 (2009) 54-61). However, Poliglumex composed ofpaclitaxel conjugated to poly(glutamic acid) currently in clinical phaseIII is strongly cationic but it was known to be more accumulated inother organs than in tumor tissue, which delays its commercialization(S. Wallace; C. Li, Adv. Drug Deliv. Rev. 60 (2008) 886-898).

PRIOR ART DOCUMENT

[Non patent document 1]

-   R. Haag, F. Kratz, Angew. Chem. Int. Ed. 45(2006)1198-1215)

DISCLOSURE Technical Problem

Therefore, it is an object of the present invention to provide a novelclass of cationic polyphosphazene compounds for drug delivery and theiranticancer drug conjugate compounds with excellent tumor selectivity andeasy drug releasing properties in the tumor site, and a preparationmethod thereof.

The present inventors have been searching clinically more efficient drugdelivery systems affording excellent tumor selectivity and easy drugreleasing properties under the above-mentioned technical background andfinally discovered that polyphosphazenes grafted with a hydrophilic PEGas solubilizing group and one selected from a multifunctional aminoacid, an oligopeptide involving amino acid or linear amino alcohol asspacer group to conjugate with an hydrophobic anticancer drug exhibitexcellent tumor selectivity due to their cationic properties and longblood circulation. The present inventors have further found that whenthe hydrophobic anticancer drugs are conjugated to the above-mentionedpolyphosphazene carrier polymer by using an acid cleavable linker, smartpolymeric anticancer drugs with excellent tumor selectivity andcontrolled releasing properties in the tumor site by acid degradationare obtained.

Technical Solution

In order to accomplish the above task, the present invention providesthe linear polyphosphazenes represented by the following chemicalformula 1.

wherein n is an integer from 1 to 300; MPEG represents methoxypoly(ethylene glycol) with a molecular weight of 350 to 1000; S is aspace group selected from the group consisting of lysine, arginine,glutamine, asparagine, tyrosine, lysine containing oligopeptide,arginine containing oligopeptide, glutamine containing oligopeptide,asparagine containing oligopeptide, tyrosine containing oligopeptide,amino-ethanol, amino-propanol, amino-butanol, amino-pentanol, andamino-hexanol; 1 equals to 0˜0.9; m equals to 0.1˜1 and 1+m equals to 1.

The present invention also provides the polyphosphazene-anticancer drugconjugates represented by the following chemical formula 2.

wherein n is an integer from 1 to 300; MPEG represents methoxypoly(ethylene glycol) with a molecular weight of 350 to 1000; S is aspace group selected from the group consisting of lysine, arginine,glutamine, asparagine, tyrosine, lysine containing oligopeptide,arginine containing oligopeptide, glutamine containing oligopeptide,asparagine containing oligopeptide, tyrosine containing oligopeptide,amino-ethanol, amino-propanol, amino-butanol, amino-pentanol, andamino-hexanol; L is a linker to connect the spacer group of the polymerand drug molecule D bearing hydroxyl or amine group; x and y areindependently in the range of 0˜0.5; z is in the range of 0˜1; andx+y+z=1.

Also, the present invention provides a method of preparingpolyphosphazene-drug conjugate compounds represented by the chemicalformula 2 comprising the steps (a) to (d):

-   (a) preparing a PEGylated polyphosphazene intermediate by subjecting    a starting material hexachlorocyclotriphosphazene to thermal    polymerization to obtain a linear poly(dichlorophosphazene) and then    reacting the linear poly(dichlorophosphazene) with the sodium salt    of methoxy poly(ethylene glycol),-   (b) preparing a hydrophilic cationic polyphosphazene carrier polymer    by reacting the PEGylated polyphosphazene intermediate with a space    group selected from the group consisting of lysine ester, lysine    containing oligopeptide ester, amino-ethanol, amino-propanol,    amino-butanol, amino-pentanol, and amino-hexanol,-   (c) preparing a drug-linker precursor by reacting a drug molecules    bearing OH or NH₂ functional group with an appropriate linker,-   (d) connecting the drug-linker precursor obtained from the Step (c)    to the space group of the hydrophilic cationic polyphosphazene    carrier polymer obtained from the Step (b).

Alternatively, the present invention provides a method of preparingpolyphosphazene-drug conjugate compounds represented by the followingchemical formula 2 comprising the steps (a) to (d):

-   (a) preparing a PEGylated polyphosphazene intermediate by subjecting    a starting material hexachlorocyclotriphosphazene to thermal    polymerization to obtain a linear poly(dichlorophosphazene) and then    reacting the linear poly(dichlorophosphazene) with the sodium salt    of methoxy poly(ethylene glycol),-   (b) preparing a hydrophilic cationic polyphosphazene carrier polymer    by reacting the PEGylated polyphosphazene intermediate with a space    group selected from the group consisting of lysine ester, lysine    containing oligopeptide ester, amino-ethanol, amino-propanol,    amino-butanol, amino-pentanol, and amino-hexanol,-   (c) connecting a linker group to the space group of the hydrophilic    cationic polyphosphazene carrier polymer obtained from the step (b),-   (d) connecting a drug molecules bearing OH or NH₂ functional group    to the linker group which is connected to the hydrophilic cationic    polyphosphazene carrier polymer obtained from the step (c).

Advantageous Effects

The cationic linear polyphosphazene compounds of the present inventionexhibit high tumor selectivity, and the linear polyphosphazene-drugconjugate compounds of the present invention were found to beaccumulated in the tumor tissues with much higher selectivity comparedwith that in other major organs such as liver and kidney in contrast tothe conventional organic polymer-drug conjugates. Furthermore, thepresent linear polyphosphazene-drug conjugate compounds are stablewithout releasing the conjugated drug in neutral blood and organs buteasily releasing in acidic tumor microenvironment, resulting in maximumdrug efficacy and minimum toxicity. Thus the cationic linearpolyphosphazene compounds and their anticancer drug conjugate compoundsof the present invention are highly promising new materials forcommercialization.

DESCRIPTION OF DRAWINGS

FIG. 1 shows particle size distribution of a cationic polyphosphazenecompound of Example 1. (The mean diameter=3.0 nm)

FIG. 2 shows the zeta potential measured for the cationicpolyphosphazene compound of Example 1.

FIG. 3 shows the particle size distribution of thepolyphosphazene-docetaxel conjugate of Example 14 (The mean diameter=60nm).

FIG. 4 shows the critical micelle concentration (CMC) of thepolyphosphazene-docetaxel conjugate of Example 14 measured by thefluorescence pyrene method.

FIG. 5 shows the time dependent degradation of thepolyphosphazene-paclitaxel conjugate of Example 20 at acidic pH 5.4 andneutral pH 7.4.

FIG. 6 (a) shows Ex vivo NIR fluorescence images of the time dependent(12 h, 24 h, 48 h, 72 h post injection) biodistributions of theCy-labeled polyphosphazene-docetaxel conjugate of Example 14 in majororgans (1: liver; 2: lung; 3: kidney; 4: spleen; 5: tumor; 6: muscle) ofthe mice inoculated with non-small cell lung tumor cells A549, (b) showsthe time dependent NIR fluorescence images of blood (WB) and plasma(PL).

FIG. 7 shows the time dependent (24 h and 48 h post injection)biodistributions in major organs (1: liver; 2: lung; 3: spleen; 4:kidney; 5: tumor; 6: heart) of Cy-labeled polyphosphazene-docetaxelconjugate of Example 14 which is IV injected to the mice inoculated withSCC7 tumor cells.

FIG. 8 shows the time dependent fluorescence intensity of each organ ofthe mice treated in FIG. 7 which is compared with that of each organ ofthe mice untreated with drug.

FIG. 9 shows mean plasma concentration-time profiles of docetaxel afterIV injection among the results of the pharmacokinetic study of thepolyphosphazene-docetaxel conjugate of Example 14 (▪) and Taxotere (♦)as reference using Sprague-Dawley rat.

FIG. 10 shows results of the xenograft trials of thepolyphosphazene-docetaxel conjugate of Example 14 using BALB/C nudemouse and MKN-28 gastric tumor cell line.

FIG. 11 shows the mouse body weight changes during the period ofxenograft trials (40 days) in FIG. 10.

FIG. 12 shows results of the xenograft trials of thepolyphosphazene-docetaxel conjugate of Example 14 using BALB/C nudemouse and non-small cell carcinoma A549 cell line.

BEST MODE

The constitution and more detailed action of the present invention areprovided. The present invention is embodied in the following descriptionbut is not limited thereto. The detailed constitution and action of thepresent invention are exemplified as in the following. In order toaccomplish the afore-mentioned purpose polyphosphazene compoundsrepresented by the following chemical formula 1 is provided.

wherein n is an integer from 1 to 300; MPEG represents methoxypoly(ethylene glycol) with a molecular weight of 350 to 1000; S is aspace group selected from the group consisting of lysine, arginine,glutamine, asparagine, tyrosine, lysine containing oligopeptide,arginine containing oligopeptide, glutamine containing oligopeptide,asparagine containing oligopeptide, tyrosine containing oligopeptide,amino-ethanol, amino-propanol, amino-butanol, amino-pentanol, andamino-hexanol; 1 equals to 0˜0.9; m equals to 0.1˜1 and 1+m equals to 1.

The above-mentioned polyphosphazene compounds of the present inventioninvolve a hydrophilic and multifunctional lysine or lysine-containinghydrophilic oligopeptide as a side group along with the hydrophilic MPEGas another side group exhibit a lower critical solution temperature(LCST) above 100° C., which is far above the body temperature incontrast to the conventional amphiphilic polyphosphazenes showing a muchlower LCST near the body temperature. Furthermore, the polyphosphazenecompounds of the present invention exhibit remarkably extended bloodhalf-life with lower systemic toxicity, and more surprising is that thepolyphosphazene compounds themselves clearly show high tumor targetingproperties probably due to their cationic properties and long bloodcirculation despite their small particle sizes (mean diameter <6 nm).The representative example of a hydrophilic oligopeptide is glycyllysine.

The present invention also furnishes the polyphosphazene-anticancer drugconjugates represented by the following chemical formula 2.

wherein n is an integer from 1 to 300; MPEG represents methoxypoly(ethylene glycol) with a molecular weight of 350 to 1000; S is aspace group selected from the group consisting of lysine, arginine,glutamine, asparagine, tyrosine, lysine containing oligopeptide,arginine containing oligopeptide, glutamine containing oligopeptide,asparagine containing oligopeptide, tyrosine containing oligopeptide,amino-ethanol, amino-propanol, amino-butanol, amino-pentanol, andamino-hexanol; L is a linker to connect the spacer group of the polymerand drug molecule D bearing hydroxyl or amine group; x and y areindependently in the range of 0˜0.5; z is in the range of 0˜1; andx+y+z=1.

For embodiment of the present invention, S represents a lysine or lysinecontaining dipeptide or tripeptide but is not limited thereto. Foranother embodiment of the present invention, S represents anamino-ethanol and amino-propanol but is not limited thereto. The drugmolecule D is desired to be hydrophobic anticancer agents such asdocetaxel, paclitaxel, camptothecin and(trans-(±)-1,2-diaminocyclohexane)platinum(II) but is not limitedthereto. The embodiment of the present invention according to theabove-mentioned chemical formula 2 may be represented by one of thefollowing chemical formula 3 to 5.

In chemical formula 3 and 4, n is independently an integer from 3 to300; MPEG represents methoxy poly(ethylene glycol) with a molecularweight of 350 to 1000; D represents docetaxel, paclitaxel, camptothecin,or (trans-(±)-1,2-diaminocyclohexane)platinum(II); R is a C₁₋₆ linear,branched or cyclic alkyl group or OCH₂Bz; x and y are independently inthe range of 0 to 0.5; z is larger than 0 and less than 1.0; x+y+z=1.

Wherein n is an integer from 3 to 300; MPEG represents methoxypoly(ethylene glycol) with a molecular weight of 350 to 1000; Drepresents docetaxel, paclitaxel, or camptothecin; R′ represents t-Bocor CBZ; x and y are independently in the range of 0 to 0.5; z is largerthan 0 and less than 1.0; x+y+z=1.

The constitutional components of the present invention are explained inthe following.

[Polyphosphazene Carrier Compounds]

The drug carrier polyphosphazene compounds of the present invention area unique inorganic/organic hybrid polymer composed of inorganic polymerbackbone consisting of alternating nitrogen and phosphorus atoms,grafted with two organic groups, one hydrophilic poly(ethylene glycol)for long blood circulation and another hydrophilic multifunctionallysine, lysine containing oligopeptide, or linear amino-alcohol as aspace group for conjugation with hydrophobic anticancer drugs. Thepresent inventors have discovered that the lysine group or lysinecontaining peptides grafted to the polyphosphazene backbone can affordcationic properties of the polyphosphazene compounds depending on thepKa value of the amino acid, which can be controlled by the amino acidemployed as spacer group. The poly(ethylene glycol) employed in thisinvention is methoxy poly(ethylene glycol) with a molecular weight inthe range of 300˜2000 and its content is determined by its mole ratio tothe space group determined by x, y and x depending on the requiredproperties such as solubility, morphology and biodegradability of thefinal conjugate drug. The molecular weight of the polyphosphazenecompounds can be controlled by the number of repeating unit n but alsoby the molecular weight of PEG. Compared with the branched linearorganic polymers, the linear polyphosphazene compounds with two organicside groups of the present invention have higher molecular weight butsmaller hydration volume, resulting in higher atomic density and bettertumor selectivity.

[Anticancer Drugs]

The anticancer active drug component to be conjugated to the branchedpolyphosphazene compounds of the present invention should have at leastone functional group of hydroxyl (OH⁻) or amine (NH₂) group and moredesirably be hydrophobic anticancer drugs such as taxane, camptothecinand platinum (II) drugs but is not limited thereto. In addition to thesesmall molecular anticancer drugs any anticancer drug molecules bearingat least one hydroxyl or amine functional group can be conjugated to thepresent polyphosphazene carrier polymers using appropriate spacer andlinker system employed in the present invention. The examples of thepresent invention will be demonstrated to show two kinds of anticancerdrugs which exhibit lowered reactivity by steric hindrance.

The afore-mentioned polyphosphazene-taxane conjugate compounds preparedby chemical conjugation of the hydrophobic taxane drug molecules to thehydrophilic polyphosphazene carrier polymer are a new class of polymericprodrug for intravenous injection, and their molecular weight can becontrolled from 3000 to 300,000 Da, but we have discovered that thefraction of 30,000 to 100,000 Da was optimum for biocompatibility andefficacy of the conjugate drugs. The hydrophilic polyphosphazene carrierpolymers of the present invention cannot form polymeric micelles inaqueous media but their conjugate chemically bound by hydrophobic taxanemolecules were found to self-assemble into strong polymeric micelleswith a mean diameter in the range of 20-100 nm, which exhibitoutstanding tumor targeting properties by EPR effect along with longblood circulation due to the PEG outer shell of the micelles, asabove-mentioned. Finally, for maximum drug efficacy along with minimumsystemic toxicity of the polyphosphazene-taxane conjugate, an acid(pH=4˜7) cleavable linker, aconitic anhydride was employed to link theanticancer drug component taxane molecules to the carrierpolyphosphazene so that the strongly cytotoxic taxane drug molecules arenot significantly released from the polyphosphazene-taxane conjugateduring their circulation in the neutral blood system (pH=7.2) but easilyreleased at the tumor site which is in the acidic tumormicroenvironment.

The docetaxel drug molecule, as displayed in the following chemicalformula 6, has four hydroxyl groups at 2, 7, 10, and 2′ positions, but2′ hydroxyl group is known to be most active, and therefore, the drugmolecule was linked to the carboxylic acid group of the linker aconiticanhydride by esterification to make a precursor, which is then reactedto form an amide bond with the amine group of the spacer group aminoacid or amino-ethanol of the polyphosphazene carrier polymer. In case ofpaclitaxel the same reaction scheme may be applied. It should be pointedout here two important reasons for employment of a multifunctionalaconitic anhydride as a linker for conjugation of taxane drug moleculesto the polyphosphazene compounds of the present invention. It is wellknown that when sterically hindered drug molecules are introduced forconjugation to the polymeric drug carriers by esterification, theproduct yield is not only very low but also sterically different isomersmay be resulted. It was found from the preliminary study that whendocetaxel was directly introduced to the aconitic anhydride linked tothe polyphosphazene carrier polymers not only long reaction time over 24hours were required but also a few sterically different isomers wereresulted. However, the present inventors have discovered that whendocetaxel was reacted first with the linker aconitic anhydride toprepare a precursor, which was reacted with the polyphosphazene carrierpolymers of the present invention, both high product yield and purity ofthe final polyphosphazene-docetaxel conjugate could be accomplished.

The present invention involves camptothecin series of the followingexemplified compounds but is not limited thereto, including all thepharmaceutically active derivatives, particularly irinotecan, topotecan,and belotecan.

[Spacer (S)]

In the present invention a multifunctional space group is introduced tothe polyphosphazene carrier polymer to chemically connect them to thelinker aconitic anhydride for conjugation with anticancer drugs. Thespacer group should contain a primary amine group to connect to theabove-mentioned linker group and another primary amine or alcoholicgroup for grafting to the polyphosphazene backbone and is one selectedfrom the multifunctional amino acid groups of lysine, arginine,glutamine, asparagine, tyrosine, and these amino acid containingoligopeptides or linear amino-alcohols. The afore-mentioned amino acidsare classified as basic amino acids, and when such a basic amino acid isintroduced to the polyphosphazene backbone, cationic properties can beafforded to the polyphosphazene compounds in a specific pH rangedepending on its pKa value of the amino acid. The space group may be notonly a single amino acid but also a combination of more than two aminoacids. For example, glycyl lysine (Gly-Lys), alanyl lysine (Ala-Lys), ora tripeptide may be employed. Furthermore, for a certain multifunctionalamino acid or peptide, the carboxylic ester group may be hydrolyzed fordirect conjugation with drug molecules without linker. Such arepresentative example is lysine, which will be illustrated in Examples.

[Linker]

The drug efficacy of the polymeric drug conjugates is well known to becritically dependent on the releasing kinetics of the active drugmolecules from the polymeric carrier. In the present invention theactive drug molecules were conjugated to the polyphosphazene carrierpolymers by either amide bonding (—CONH—) or ester boding (—COO—) byappropriate selections of the space group and the linker aconiticanhydride depending on the molecular structures of drug and carriersystems, which is different from the conventional method useful only forthe drug molecules bearing a primary amine group. Thus the presentinvention furnishes a new and upgraded spacer-linker system forintroduction of acid-cleavable linker for drug conjugation.

The afore-mentioned conjugation reactions may be performed under generalcoupling reaction conditions in any organic solvents inert to theconjugation reactions including dichloromethane, chloroform,acetonitrile, 1,4-dioxane, dimethyl formamide, and tetrahyrofuran.However, because of instability of the reaction intermediates to beformed during introduction of the linker, the whole reactions wereperformed in dried solvents and under dried argon atmosphere at lowtemperature (−10˜0° C.) to increase reaction efficiency. In particular,cis-aconitic anhydride linker is known to undergo its anhydride ringopening reaction in basic solution, resulting in isomers, and therefore,instead of the basic catalyst DMAP (dimethylaminopyridine), DPTS(dimethylaminopyridine/p-toluene sulfonic acid) was employed as acatalyst to perform the reaction in neutral state. The representativedrugs appropriate for preparation of the polyphosphazene conjugatecompounds are oligopeptides, polypeptides, monomeric drugs, antibody,nucleotides, lipids, and any other materials bearing —OH or NH₂functional group.

The linkers usable in the present invention are listed in the followingchemical formula 7 to 11 but chemical formula 7 is most preferred.

In case of acid cleavable (pH=5.5) aconitic anhydride of the abovechemical formula 7 employed as a linker the polymer conjugate drugs maybe prepared according to the following reaction scheme 1.

However, in this reaction scheme an inactive isomer unable to releasethe drug moiety is formed, and depending on the conjugation site, drugreleasing rate and drug efficacy is difficult to control. In particular,in case of both the spacer/linker and linker/drug bonds are amide bond,an isomer (Reaction scheme 1 Inactive) difficult to release drugmolecules by hydrolysis may be formed, resulting in low drug efficacy.In case of Reaction scheme 2 showing both spacer/linker and linker/drugbonds are ester bond, also inactive form may be resulted althoughhydrolysis by enzymes may be workable. Also, in case of the anhydridering opening by using the OH group of drug molecule, nucleophilic agentshould be used in large excess particularly for sterically hindereddrug. In case of taxane drugs of the present invention approximately 20times excess amount was required to complete the reaction. In such case,the product yield of the final polyphosphazene-drug conjugate was foundto be less than 5%, which is not commercially feasible.

In contrast to the above reaction schemes 1 and 2 starting from theanhydride ring opening reaction of the aconitic anhydride, the followingreaction scheme 3 discovered in the present invention shows that insteadof the aconitic anhydride ring opening reaction its carboxylic group wasreacted with the hydroxyl group of a drug molecule (HOR′) to form anester bond yielding a drug precursor. This precursor was then conjugatedto the drug carrier polymer (RNH₂) by the anhydride ring openingreaction using the spacer amine group of the carrier polymer. Theaconitic anhydride ring opening reaction also can be performed usingexcess NHS(N-hydroxy succinimide) as shown in the above reaction scheme.This synthetic route gives important advantages over the afore-mentionedmethods. First of all, inactive isomers shown in the above Reactionschemes 1 and 2 can be avoided, resulting in much higher yield of thefinal conjugate drug, and the purity of the conjugate drug as well asdrug efficacy could be reasonably controlled.

The present invention also provides detailed synthetic methods of thelinear polyphosphazene compounds and polyphosphazene-drug conjugatecompounds. In this regard, the concrete examples of the drug, spacer andlinker groups will be demonstrated, but the present invention is notlimited to such examples.

The linear polyphosphazene compounds of the present invention issynthesized by grafting a hydrophilic poly(ethylene glycol) (PEG) and amultifunctional lysine bearing two primary amine and one carboxylgroups, lysine containing oligopeptides or amino-ethanol to thedichlorophosphazene backbone (—N═PCl₂—). The multifunctional lysine,lysine containing oligopeptides or linear amino-alcohol of thus preparedpolyphosphazene compounds may be used as a space group to conjugate withhydrophobic anticancer drugs such as taxane, platinum(II) complex orcamptothecin using a linker such as aconitic anhydride to produce novelacid cleavable linear polyphosphazene-drug conjugates.

The linear polyphosphazene-drug conjugate compounds of the presentinvention exhibited outstanding tumor selectivity by selectiveaccumulation in tumor tissue, and it was found that by controlling thex, y, and z value of the afore-mentioned chemical formula 2 highly watersoluble, long blood circulating and excellent tumor targeting anticancerdrugs could be prepared.

In the present invention the spacer group selected from theafore-mentioned lysine, arginine, glutamine, asparagine, or tyrosinecontaining oligopeptides is desirably lysine and glycine containingoligopeptide, for example, dipeptide and tripeptide and more desirablyglycyllysine but is not limited thereto. In the afore-mentionedoligopeptides lysine is desired to be located at the terminal positionto conjugate with taxane anticancer drug. The space group selected fromthe linear amino-alcohols is desirably amino-ethanol, amino-propanol,amino-butanol, amino-pentanol and amino-heanol, and more desirablyamino-ethanol and amino-propanol.

In the present invention the taxane anticancer drug (D) molecules areconjugated to the polyphosphazene carrier polymer through the lysineamine or carboxyl group, and in case the carboxyl group is used forconjugation, the amine group should be blocked by a blocking group suchas t-Boc, FMOC, or CBZ. In the afore-mentioned chemical formula 2, L isa linker to connect the anticancer drug (D) and spacer group of thecarrier polymer (S) and is cis-aconitic anhydride, succinyl anhydride,or maleic anhydride and desirably cis-aconitic anhydride. The anticancerdrug components (D) exemplified in the present invention are taxanefamily, camptothecin family, and platinum complexes including docetaxel,paclitaxel, camptothecin, topotecan, irinotecan, belotecan, oxaliplatin,but is not limited to these drugs.

The polyphosphazene-drug conjugates of the present invention have anarrow molecular weight distribution in the range of 3,000-300,000 anddesirably in the range of 30,000-100,000 and are highly soluble in waterby self-assembling into polymeric micelles with a mean diameter in therange of 20˜200 nm depending on the hydrophobicity of the drug molecules(D). As above-mentioned, such polymeric micelles assembled from thepolyphosphazene-drug conjugates of the present invention exhibitexcellent tumor selectivity attributed to their EPR effect.

The polyphosphazene and polyphosphazene-taxane conjugate compounds canbe prepared by the following four step synthetic reactions.

-   -   (a) The starting material hexachlorocyclotriphosphazene is        converted by thermal polymerization into linear        poly(dichlorophosphazene), (N═PCl₂)_(n), which is then reacted        with sodium salt of poly(ethylene glycol) to obtain an        intermediate polyphosphazene, [N═PCl(MPEG)]n;    -   (b) The above polyphosphazene intermediate is reacted with one        selected from the group consisting of lysine ester, lysine        containing oligopeptide, amino-ethanol, amino-propanol,        amino-butanol, amino-penanol, and amino-hexanol to prepare        hydrophilic cationic polyphsphazene carrier polymers;    -   (c) An anticancer drug bearing hydroxyl (OH) or primary amine        (NH₂) group is reacted with a linker group, for example,        aconitic anhydride, to prepare a precursor; and    -   (d) Finally, the precursor of step (c) is reacted with the        polyphsphazene carrier polymer of step (b) to obtain the final        polyphosphazene conjugate compound of chemical formula 2.

wherein n is an integer from 1 to 300; MPEG represents methylpoly(ethylene glycol) with a molecular weight of 350 to 1000; S is aspace group selected from the group consisting of lysine, arginine,glutamine, asparagine, tyrosine, lysine containing oligopeptide,arginine containing oligopeptide, glutamine containing oligopeptide,asparagine containing oligopeptide, tyrosine containing oligopeptide,amino-ethanol, amino-propanol, amino-butanol, amino-pentanol, andamino-hexanol; L is a linker to connect the spacer group of the polymerand drug molecule D bearing hydroxyl or amine group; x and y areindependently in the range of 0˜0.5; z is in the range of 0˜1; andx+y+z=1.

In another embodiment of the present invention, the anticancer drugbearing hydroxyl (OH) or primary amine (NH₂) group can be one selectedfrom the group of docetaxel, paclitaxel, camptothecin, and(trans-(±)-1,2-diaminocyclohexane)platinum(II), but is not limited tothese drugs.

Furthermore, the abovementioned four stepwise reactions to preparepolyphosphazene-drug conjugates may be subjected to a littlemodification depending on the structure of drug molecules to beconjugated. For example, in case of platinum(II) complex, instead ofpreparation of the precursor by reaction of a drug with the linker atstep (c) followed by reaction of the precursor to the polymer carrier atstep (d), the linker group is reacted to the polymer carrier at step(c), and then drug is linked to the linker group connected to thecarrier polymer at step (D).

All the following synthetic reaction procedures should be performedunder carefully controlled inert atmosphere using dried argon ornitrogen and thoroughly dried solvents. Detailed synthetic proceduresare described in the following.

Step (a)

Hexachlorocyclotriphosphazene, (N═PCl₂)₃, shown in chemical formula 12was subjected to thermal polymerization according to the literatureprocedure (Youn Soo Sohn, et al. Macromolecules, 1995, 28, 7566) toobtain poly(dichlorophosphazene) (N═PCl₂)_(n) with an average molecularweight of 10⁴˜10⁵ as represented in chemical formula 13.

Wherein n represents the degree of polymerization in the range of 3 to300.

The monomethoxy poly(ethylene glycol) of chemical formula 14 was driedby using azeotropic mixture of toluene and water and then reacted withsodium metal to convert to sodium salt of chemical formula 15, which wasthen reacted with poly(dichlorophosphazene) of chemical formula 13 inthe presence of triethylamine to complete PEGylation of polyphosphazeneof chemical formula 15.

In the above chemical formula 14 and 15 a is the degree ofpolymerization of PEG and is 7 to 22.

More detailed procedure for preparation of PEGylated polyphosphazene isdescribed in the following.

The hexachlorocyclotriphosphazene (N═PCl₂)₃ of chemical formula 12 and 3to 10% anhydrous aluminum chloride are mixed homogeneously in the glovebox and then sealed in a Pyrex ample under vacuum. The ample was heatedto 230-250° C. with rotation in a heating chamber for 3-5 h to obtain aclear viscous liquid of poly(dichlorophosphazene). In the meantime,monomethoxy poly(ethylene glycol) of chemical formula 14 is reacted with1.2-1.5 equivalent sodium metal in an unreactive anhydrous organicsolvent such as tetrahydrofuran (THF), benzene and toluene to obtain asodium salt of chemical formula 15. To a solution of one molarpoly(dichlorophosphazene) of chemical formula 13 dissolved in the samesolvent was added 0.5-1.8 equivalent of the sodium salt of chemicalformula 15 above prepared. The reaction solvent may be any unreactivesolvent but desirably tetrahydrofuran, benzene, toluene and chloroform.The sodium salt solution of chemical formula 15 should be added slowlyfor 2-8 h to the poly(dichlorophosphazene) solution cooled to below 0°C., and then the reaction mixture was further reacted at ambienttemperature for 6-24 h to obtain the PEGylated polyphosphazeneintermediate of chemical formula 16.

Wherein n is the degree of polymerization of polyphosphazene in therange of 3 to 300, a is the degree of polymerization of methoxypoly(ethylene glycol) in the range of 7 to 22, and b is the substitutedmole fraction of methoxy poly(ethylene glycol) in the range of 0.5 to1.8.

Step (b)

In order to substitute the rest (2-b) chlorine atoms of the PEGylatedpolyphosphazene of chemical formula 16 with a space group for drugconjugation, 1.5-1.8 equivalent of lysine ester or lysine containingoligopeptide ester dissolved along with 6 equivalent triethylamine inany unreactive organic solvent, desirably, tetrahydrofuran, chloroform,or dichloromethane is slowly added to the above PEGylatedpolyphosphazene intermediate solution of chemical formula 16 and thenrefluxed at 40-60° C. for 12 h to 3 days. As a lysine ester the lysinederivatives of chemical formula 17 may be used and as a lysinecontaining oligopeptide ester the derivatives of chemical formula 18 maybe used. In the chemical formula glycine may be substituted by leucine,isoleucine, phenyl alanine, and valine.

Wherein R is linear, branched, or cyclic C₁₋₆ alkyl group, or OCH₂Bz,and R′ is amine protecting group, t-Boc (tert-butoxycarbonyl), Fmoc(fluorenylmethyloxycarbonyl) or CBZ (carbozenyloxy).

Wherein R is a linear, branched, or cyclic C₁₋₆ alkyl group, or OCH₂Bz,and R′ is an amine protecting group, t-Boc (tert-butoxycarbonyl), Fmoc(fluorenylmethyloxycarbonyl) or CBZ (carbozenyloxy).

In the present invention, R may be methyl, ethyl, n-propyl, n-butyl ort-butyl, but is not limited to them.

The above-mentioned reaction solution was centrifuged or filtered toremove the byproduct precipitate (Et₃NHCl or NaCl) and the filtrate wassubjected to concentration. Ethanol was added to the concentratefollowed by vacuum concentration to remove organic solvents completely.The oily product was dissolved in a small amount of ethanol (100 ml) andthen a large amount of water (900 ml) was added for recrystallization atlow temperature, and then subjected to membrane ultrafiltration usingmembranes with different pore sizes to fractionate the polymers intomolecular weight of 50,000 to 100,000. The fractionated polyphosphazenesolution was subjected to freeze-dry to obtain polyphosphazene compoundsof chemical formula 19 and chemical formula 20 in approximately 40-50%yield.

In chemical formula 19 and 20, n is the degree of polymerization ofpolyphosphazenes ranging in 3 to 300; MPEG represents methoxypoly(ethylene glycol) with a molecular weight of 350 to 1000; b has avalue of 0.5-1.8; R is a linear, branched, or cyclic C₁₋₆ alkyl group,or OCH₂Bz; and R′ is an amine protecting group, t-Boc(tert-butoxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl) or CBZ(carbozenyloxy).

Step (c)

It is not difficult to conjugate a drug molecule with a functional groupto the polyphosphazene compounds bearing a multifunctional space groupprepared at Step (b), but it is not easy to conjugate a drug moleculebearing multifunctional groups such as taxane to the multifunctionalpolyphosphazene carrier polymer in a desired bonding mode which isclinically useful. In other words, the anticancer drug-polyphosphazeneconjugate is required to exhibit strong tumor targeting properties anddrug releasing kinetics in addition to satisfactory physicochemicalproperties. In particular, the drug releasing kinetics of the polymerconjugate is known to be critically important for drug efficacy, andtherefore, the role of linker of the present invention is veryimportant. In more concrete, the linker group should not only be able toconnect easily the functional group of drug molecule (OH, NH₂) to thefunctional groups of the polyphosphazene carrier polymer (COOH, NH₂),but also be able to let the conjugate drug to release easily drugmolecules in the targeted tumor site.

It has been discovered in the present invention that cis-aconiticanhydride of chemical formula 7 is the best linker for conjugation oftaxane to the polyphosphazene carrier polymer in both aspects ofsynthetic and drug releasing kinetics, particularly when the linker isreacted first with taxane to prepare a precursor, which is then linkedto the space group of polyphosphazenes. The taxane drug precursor isprepared as in the following.

Docetaxel (1.0 mmol, 803 mg) and cis-aconitic acid anhydride (2.0 mmol,312 mg) are mixed under argon atmosphere and dissolved in THF ordichloromethane. The mixed solution is cooled to the freezing point andthen DIC (N,N′-diisopropylcarbodiimide) (2.0 mmol, 0.252 g) and DPTS(4-(N,N′-dimethylamino)pyridinium-4-toluene sulfonate) (2.0 mmol, 0.58g) are added to perform esterification reaction between carboxylic acidgroup of the linker aconitic acid and 2′-hydroxy group of docetaxel for12 h. After confirming the completion of the reaction by TLC, excessamounts of NHS (N-hydroxy succinimide) and DIPEA (diisopropylethylamine)are added to open the anhydride ring of aconitic acid unreacted. Theaconitic anhydride ring is known to be easily opened by hydroxyl andamine group in basic solution. After stirring for 12 h the reactionmixture is cooled to 0° C. for 3 h, and then vacuum filtered to removeprecipitate. The filtrate is subjected to vacuum distillation to removeall the organic solvents to obtain an oily mixture, which is dissolvedin ethanol (50 ml). A large excess amount of water (500 ml) is added tothis ethanol solution for recrystallization for 3 h in refrigerator. Thesupernatant liquid is removed to obtain an oily product which iscompletely dissolved in 300 ml dichloromethane. This dichloromethanesolution is washed with saturated salt solution three times and then thecollected organic layer is dried using anhydrous NaHCO₃ and thensubjected to vacuum evaporation to obtain the precursor in 95% yield.

Step (d)

To the afore-mentioned polyphospazene carrier polymer prepared at step(b) a hydrophobic anticancer drug such as taxane is conjugated to obtaina novel polyphosphazene-taxane conjugate represented by chemical formula2. There are two different ways to conjugate taxane molecules to thepolyphosphazene carrier polymer: one is that the taxane molecule islinked via aconitic acid to the lysine amine of the polyphosphazenecarrier by amide bonding and another is esterification of the lysinecarboxyl group of the polyphosphazene carrier with 2′-hydroxyl group ofthe taxane molecule. Therefore, the step (d) is performed in twodifferent methods.

In the amide bonding method, the protecting group (t-Boc) of theafore-mentioned polyphosphazene carrier polymer of chemical formula 19or chemical formula 20 should be removed by reaction with a mixture oftrifluoroacetic acid and methylene chloride (2:1) for 6 h followed byneutralization, washing with water and then freeze-drying. Such anunblocked polyphosphazene carrier polymer is reacted for 12 h with adrug precursor, aconitic taxane-NHS (N-hydroxysuccinimidyl) prepared byreaction of the linker aconitic anhydride with the drug such as taxanefollowed by addition of excess NHS (N-hydroxysuccinimide) and DIPEA(diisopropylethylamine). The resultant reaction mixture is subjected tovacuum evaporation for concentration for 12 h and then dissolved inethanol. The final solution is subjected vacuum evaporation (37° C., 5mm bar) to remove all the trace of solvents and DIPEA. The residualpolymer conjugate product is dissolved in 50 ml of ethanol and then 950ml of distilled water is added to the ethanol solution forrecrystallization in refrigerator. After 3 h the solution mixture isvacuum filtered to collect the final polymer conjugate product, which iswashed several times with 30% ethanol solution and then distilled waterusing ultra-membrane until less than 0.1% unreacted taxane is detectedby UV spectroscopy. The purified polyphosphazene-taxane conjugatesolution is freeze dried to obtain the final product of chemical formula3 or 4.

In the ester bonding method, the terminal lysine ester of the spacergroup of polyphosphazene carrier polymer can be hydrolyzed to carboxylicacid form using an alkali and then esterification with the 2′-hydroxylgroup of taxane can be performed. For example, the polyphosphazenecarrier polymer of chemical formula 19 is dissolved in methanol and thenexcess amount of KOH or NaOH (150˜200%) is added thereto to obtain thepotassium or sodium salt of lysine of the carrier polymer. The methanolsolvent is removed by vacuum evaporation and the resultant metal salt isdissolved in distilled water (100 ml). Methylene chloride or chloroform(300 ml) is added to this solution, which is acidified by slow additionof an organic acid. When the pH of the water layer is lowered down to4-3, the polyphosphazene carrier polymer in acidic form is extractedinto the organic layer by shaking the two solvent layers. Suchextraction procedure is repeated three times and the collected organiclayer solution is dried by anhydrous sodium bicarbonate. Filtration andvacuum evaporation of the dried solution gives a polyphosphazene carrierpolymer bearing terminal hydrolyzed lysine group. The resultingpolyphosphazene carrier polymer bearing hydrolyzed lysine and equivalentdocetaxel are dissolved in tetrahydrofuran and thenN,N′dicyclohexylcarbodiimide and triethylamine are added thereto foresterification reaction. Completion of the reaction is confirmed by TLCusing a solvent mixture (CHCl₃:MeOH₃=10:1), and then the reactionmixture is subjected to vacuum distillation to concentrate the reactionmixture, followed by ultra-membrane fractionation of the molecularweight of 30 to 100 kDa of the final polyphosphazene-docetaxel conjugateof chemical formula 5.

Hereinafter the constitution and action of the present invention will bedescribed in more detail with reference to the following examples anddemonstrations, but the present invention is not limited thereto. In thefollowing examples, elemental analysis of carbon, hydrogen and nitrogenfor the compounds of the present invention was performed usingPerkin-Elmer C, H and N analyzer. Hydrogen and nitrogen nuclear magneticresonance spectra were measured using Varian Gemini-500 NMRSpectrometer. The particle size distributions of polyphosphazene carrierpolymers and their drug conjugates were measured using MalvernZeta-sizer (Nano-ZS).

MODE FOR INVENTION

Synthesis of Polyphosphazene Compounds (Drug Delivery Systems)

EXAMPLE 1 Synthesis of [NP(MPEG550)_(1.5)(LysEt)_(0.5)]_(n)

Hexachlorocyclotriphosphazene ([NPCl₂]₃, 11.54 g, 100 mmol) andanhydrous aluminum chloride (AlCl₃, 7.5 wt %) are mixed homogeneously inglove box and then subjected to thermal polymerization at 250° C. for 5h according to the literature procedure (Sohn Y. S. et al.Macromolecules 1995, 28, 7566) to obtain poly(dichlorophosphazene)([NPCl₂]_(n)). In the meantime, methoxypoly(ethylene glycol) with anaverage molecular weight of 550 (MPEG550) (82.5 g, 150 mmol) is reactedwith sodium metal (4.9 g, 200.4 mmol) at 120° C. for 6 h in driedtoluene solvent under argon atmosphere to obtain sodium salt of MPEG550.To the above poly(dichlorophosphazene) dissolved in tetrahydrofuran (100ml) in a glass vessel is added slowly for 60 min the solution of sodiumsalt of MPEG550 prepared in ice bath (0° C.). After 1 h the ice bath isremoved and then the reaction mixture is subjected to further reactionat ambient temperature for 12 h to obtain PEGylated polyphosphazeneintermediate solution. In a separate vessel Boc-lysine ethyl ester(N-Boc-LysEt, 20.5 g, 75.0 mmol) neutralized by dried trimethylamine indried chloroform (200 ml) is slowly added to the above PEGylatedpolyphosphazene intermediate solution and the reaction mixture isfurther reacted at room temperature for 24 h. The reaction mixture isfiltered to remove the resultant byproduct precipitates (NEt₃.HCl orNaCl) and the filtrate is subjected to vacuum evaporation to concentratethe solution, which is dissolved in ethanol followed by vacuumevaporation. Finally the residue is dissolved in distilled water, whichis filtered to remove any insoluble, and the filtrate is subjected todialysis using ultra membrane (CE, MWCO=3000) to remove lower molecularweight fraction under 3000 Da. The fractionated polypphosphazene polymeris washed with distilled water more than 5 times and then freeze-dried.The resultant polypphosphazene polymer is dissolved in a dichloromethanesolution (200 ml) containing 30% (v/v) trifluoroacetic acid, which isstirred for 6 h to remove the blocking group (t-Boc) from the polymer toobtain the unblocked polypphosphazene polymer[NP(MPEG550)_(1.5)(LysEt)_(0.5)]_(n) in 65% yield (3.37 g). Afterconfirming the unblocked lysine amine by proton NMR, the polymersolution was neutralized by tri-ethyl amine, followed by vacuumdistillation to remove organic solvent. To the resulting polymer aqueoussodium bicarbonate solution was added to dissolve the polymercompletely, which was desalted and then fractionated usingultra-membranes with molecular weight cut-off at 3k Da, 30 k Da, 100 kDa. The overall recovery yield was 90%.

Composition: C₈₃H₁₇₀N₄O₄₁P₂

Elemental analysis data (%): C, (51.66); H, (8.59); N, (2.83).Theoretical value: (51.33); H, (8.82); N, (2.88).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.25 (s, 1.5H, Lys-OCH₂CH₃), 2.49 (br,1.00H, suucinyl-CH₂), 2.90 (br, 1.00H, Lys-ε-CH₂), 3.38 (s, 4.50H,MPEG550-OCH₃), 3.65 (br, 66.0H, MPEG550-OCH₂CH₂), 4.4 (s, 1H, Lysine-CH.

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 2 Synthesis of [NP(MPEG750)_(1.5)(LysEt)_(0.5)]_(n)

According to the same procedure as described in Example 1, the desiredproduct of the title polyphosphazene compound was prepared usinghexachlorocyclotriphosphazene ([NPCl₂]₃, 11.54 g, 100 mmol),methoxypoly(ethylene glycol) with an average molecular weight of 750(MPEG750, 112.5 g, 150 mmol), trimethylamine (80.0 ml, 600 mmol), andBoc-LysEt, (20.5 g, 75 mmol) in 74% yield.

Composition: C₁₀₇H₂₁₈N₄O₅₃P₂.

Elemental analysis data (%): C, (51.30); H, (8.99); N, (2.36).Theoretical value: (52.01); H, (8.89); N, (2.27).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.25 (s, 1.50H, Lys-OCH₂CH₃), 1.39-1.98(br, 3.00H, Lys-CH₂), 2.90 (br, 1H, Lys-ε-CH₂), 3.38 (s, 4.50H,MPEG750-OCH₃), 3.65 (br, 98.0H, MPEG750-OCH₂CH₂), 4.01 (bs, 6H,MPEG750-CH₂), 4.45 (m, 0.5H, Lys-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 3 Synthesis of [NP(MPEG1000)_(1.5)(LysEt)_(0.5)]_(n)

According to the same procedure as described in Example 1, the desiredproduct of the title polyphosphazene compound was prepared usinghexachlorocyclotriphosphazene ([NPCl₂]₃, 11.54 g, 100 mmol),methoxypoly(ethylene glycol) with an average molecular weight of 1000(MPEG1000, 150 g, 150 mmol), trimethylamine (80.0 ml, 600 mmol), andBoc-LysEt, (20.5 g, 75 mmol) in 74% yield.

Composition: C₁₄₃H₂₉₀N₄O₇₁P₂.

Elemental analysis data (%): C, (53.01); H, (8.70); N, (2.36).Theoretical value: (52.62); H, (8.96); N, (1.72).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.25 (s, 1.50H, Lys-OCH₂CH₃), 1.39-1.98(br, 3.00H, Lys-CH₂), 2.90 (br, 1H, Lys-ε-CH₂), 3.38 (s, 4.50H,MPEG1000-OCH₃), 3.65 (br, 130.0H, MPEG1000-OCH₂CH₂), 4.45 (m, 0.5H,Lys-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 4 Synthesis of [NP(MPEG550)_(1.25)(LysEt)_(0.75)]_(n)

According to the same procedure as described in Example 1, the desiredproduct of the title polyphosphazene compound was prepared usinghexachlorocyclotriphosphazene ([NPCl₂]₃, 11.54 g, 100 mmol),methoxypoly(ethylene glycol) with an average molecular weight of 550(MPEG550, 69.0 g, 126 mmol), trimethylamine (80.0 ml, 600 mmol), andBoc-LysEt, (27.4 g, 100 mmol) in 74% yield.

Composition: C_(74.5)H₁₅₃N₅O_(35.5)P₂

Elemental analysis data (%): C, (50.87); H, (9.02); N, (4.31).Theoretical value: (51.14); H, (8.82); N, (4.14).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.25 (s, 2.25H, Lys-OCH₂CH₃), 1.39-1.98(br, 4.50H, Lys-CH₂), 2.90 (br, 1.5H, Lys-ε-CH₂), 3.38 (s, 3.75H,MPEG550-OCH₃), 3.65 (br, 82.5H, MPEG550-OCH₂CH₂), 4.45 (m, 0.7H,Lys-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 5 Synthesis of [NP(MPEG550)_(1.0)(LysEt)_(1.0)]_(n)

According to the same procedure as described in Example 1, the desiredproduct of the title polyphosphazene compound was prepared usinghexachlorocyclotriphosphazene ([NPCl₂]₃, 11.54 g, 100 mmol),methoxypoly(ethylene glycol) with an average molecular weight of 550(MPEG550, 55.0 g, 100 mmol), trimethylamine (80.0 ml, 600 mmol), andBoc-LysEt, (35.6 g, 130 mmol) in 74% yield.

Composition: C₆₆H₁₃₆N₆O₃₀P₂

Elemental analysis data (%): C, (50.29); H, (8.95); N, (5.51).Theoretical value: (50.95); H, (8.81); N, (5.40).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.25 (s, 3.00H, Lys-OCH₂CH₃), 1.39-1.98(br, 6.00H, Lys-CH₂), 2.90 (br, 2.0H, Lys-ε-CH₂), 3.38 (s, 3.0H,MPEG550-OCH₃), 3.65 (br, 66.0H, MPEG550-OCH₂CH₂), 4.45 (m, 1.0H,Lys-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 6 Synthesis of [NP(MPEG550)_(1.5)(GlyLysEt)_(0.5)]_(n)

According to the same procedure as described in Example 1, the desiredproduct of the title polyphosphazene compound was prepared usinghexachlorocyclotriphosphazene ([NPCl₂]₃, 11.54 g, 100 mmol),methoxypoly(ethylene glycol) with an average molecular weight of 550(MPEG550, 82.5 g, 150 mmol), trimethylamine (80.0 ml, 600 mmol), andGly(N^(ε)—BocLysEt (24.8 g, 90 mmol) in 74% yield.

Composition: C₈₅H₁₇₃N₅O₄₂P₂

Elemental analysis data (%): C, (50.75); H, (8.82); N, (3.61).Theoretical value: (51.06); H, (8.72); N, (3.50).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.25 (s, 3.00H, Lys-OCH₂CH₃), 1.39-1.98(br, 6.00H, Lys-CH₂), 2.90 (br, 2.0H, Lys-ε-CH₂), 3.38 (s, 3.0H,MPEG750-OCH₃), 3.65 (br, 66.0H, MPEG500-OCH₂CH₂), 3.92 (bs, 2H,Gly-CH₂), 4.45 (m, 1.0H, Lys-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 7 Synthesis of [NP(MPEG550)_(1.5)(N^(α)-BocLys)_(0.5)]_(n)

According to the same procedure as described in Example 1, the esterform of the title polyphosphazene compound,[NP(MPEG550)_(1.5)(N^(α)-BocLysEt)_(0.5)] was prepared (75% yield) usinghexachlorocyclotriphosphazene ([NPCl₂]₃, 11.54 g, 100 mmol),methoxypoly(ethylene glycol) with an average molecular weight of 550(MPEG550, 82.5 g, 150 mmol), trimethylamine (80.0 ml, 600 mmol), andN^(ε)-BocLysEt (20.5 g, 75 mmol). This synthetic derivative (10 g, 10mmol) and NaOH (0.4 g, 10 mmol) were dissolved in methanol and thensubjected to hydrolysis at ambient temperature for 4 h. Completehydrolysis was confirmed by using proton NMR, and then the solution wassubjected to vacuum evaporation to obtain a solid state polymer, whichwas dissolved in distilled water and acidified to pH=3 by organic acid.This acidic polymer solution was extracted with chloroform ordichloromethane three times. The organic layer was dried using anhydroussodium bicarbonate and then subjected to vacuum evaporation to obtainthe title compound (95% yield).

Composition: C₈₆H₁₇₄N₄O₄₃P₂

Elemental analysis data (%): C, (51.02); H, (8.94); N, (2.91).Theoretical value: (51.28); H, (8.71); N, (2.78).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.32 (s, 4.5H, Boc-CH₃), 1.39-1.98 (br,6.00H, Lys-CH₂), 2.90 (br, 2.0H, Lys-ε-CH₂), 3.38 (s, 3.0H,MPEG550-OCH₃), 3.65 (br, 66.0H, MPEG550-OCH₂CH₂), 4.45 (m, 1.0H,Lys-CH).

EXAMPLE 8 Synthesis of [NP(MPEG550)(AE)]_(n)

According to the same procedure as described in Example 1, PEGylatedpolyphosphazene intermediate was prepared usinghexachlorocyclotriphosphazene ([NPCl₂]₃, 2.0 g, 5.72 mmol), thecatalytic aluminium chloride (AlCl₃, 7.0 wt %), methoxypoly(ethyleneglycol) with an average molecular weight of 550 (MPEG550, 9.48 g, 17.2mmol), and sodium metal (0.59 g, 25.7 mmol). In the meantime,2-aminoethanol (AE, 1.30 g, 21.3 mmol) and sodium hydride (0.61 g, 25.4mmol) were reacted in dried tetrahydrofuran (50 ml) at ambienttemperature for 5 h to obtain a yellow precipitate of sodium salt of2-aminoethanol, which was washed thoroughly with ethyl ether and thendissolved in dimethyl sulfoxide (50 ml). The resultant solution wasadded to the above-mentioned PEGylated polyphosphazene intermediate, andthe reaction mixture was further reacted for 24 h at 50° C. The reactionmixture was filtered to remove the byproduct sodium chloride and thefiltrate was subjected to dialysis using cellulose membrane (MWCO: 3.5kDa) and freeze drying to obtain a new polyphosphazene carrier polymerin 70% yield.

Composition: C₂₇H₅₇N₂O₁₄P.H₂O

Elemental analysis data (%): C, (47.38); H, (8.61); N, (3.95).Theoretical value: (47.45); H, (8.64); N, (4.10).

¹H-NMR spectra (CDCl₃) (δ, ppm): 3.26 (s, 3H, OCH₃ of MPEG), 3.50-3.52(m, 4H, (CH₂)₂ of aminoethanol), 3.54-3.81 (brm, 46H, CH₂ of MPEG),3.83-4.10 (brm, 2H, —P—O—CH₂— of MPEG).

³¹P-NMR spectra: (CDCl₃, ppm): δ −2.66 (O—P—O).

EXAMPLE 9 Synthesis of [NP(MPEG750)(AE)]_(n)

According to the same procedure as described in Example 8, the desiredproduct of the title polyphosphazene compound was prepared usinghexachlorocyclotriphosphazene ([NPCl₂]₃, 2.0 g, 5.72 mmol), catalyticaluminum chloride (AlCl₃, 7.0 wt %), methoxypoly(ethylene glycol) withan average molecular weight of 750 (MPEG750, 12.9 g, 17.2 mmol), sodiummetal (0.59 g, 25.7 mmol), 2-aminoethanol (1.30 g, 21.3 mmol) and sodiumhydride (0.61 g, 25.4 mmol) in 78% yield.

Composition: C₃₅H₇₃N₂O₁₈P.H₂O.

Elemental analysis data (%): C, (48.02); H, (8.96); N, (3.55).Theoretical value: (48.89); H, (8.73); N, (3.26).

¹H-NMR spectra (CDCl₃) (δ, ppm): 3.41 (s, 3H, OCH₃ of MPEG), 3.49-3.53(m, 4H, (CH₂)₂ of aminoethanol), 3.57-3.83 (brm, 62H, CH₂ of MPEG),3.83-4.05 (brm, 2H, —P—O—CH₂— of MPEG).

³¹P-NMR spectra: (CDCl₃, ppm): δ −3.95 (O—P—O).

Synthesis of Anticancer Drug-Linker Precursor

EXAMPLE 10 Synthesis of 2′-aconitic docetaxel NHS ester

Aconitic anhydride (3.12 g, 20 mmol), docetaxel (8.03 g, 10 mmol) and acatalyst DPTS (dimethylaminopyridine/p-toluenesulfonic acid) (6.0 g, 20mmol) were vacuum-dried for 4 h and cooled to −10° C. and thencompletely dissolved in dried tetrahydrofuran (100 ml). To this solutionwas slowly added DIC (N,N′-diisopropylcarbodiimide) (2.5 g, 20 mmol)dissolved also in dried tetrahydrofuran. The mixed reaction solution wasreacted for 6 h at −10° C. and then for 6 to 12 h at 0° C. The progressof the reaction was monitored by TLC using a solvent mixture ofdichloromethane:methanol (95:5) until no free docetaxel is detected.After confirmed completion of the reaction, excess amount of NHS(N-hydroxysuccinimide) (11.5 g, 100 mmol) and a large excess of basicDIPEA(diisopropyl ethyl amine) were added to the above reaction solutionand the reaction mixture was further reacted for 12 h. The reactionmixture was finally subjected to vacuum evaporation to obtain a solidpolymer product, which was dissolved in small amount of ethanol (50 ml)and then a large amount of water (950 ml) was added thereto forrecrystallization at 0° C. for 3 h. The supernatant water layer isremoved to obtain brown oily product, which was dissolved in chloroformor dichloromethane for washing successively with salt solution (pH=7),citric acid solution (pH=2), sodium bicarbonate solution (pH=9) andfinally with salt solution. The final organic solution was dried usinganhydrous magnesium sulfate and then vacuum dried to obtain finally theprecursor composed of 2′-acconitic-docetaxel-NHS ester.

Composition: C₅₃H₆₀N₂O₂₁

Elemental analysis data (%): C, (60.07); H, (5.86); N, (2.71).Theoretical value: (59.99); H, (5.70); N, (2.64).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.13 ppm (s, 3H, C17-CH₃), 1.24 ppm (s,3H, C16-CH₃), 1.34 ppm (s, 9H, C60-t Bu), 1.75 ppm (s, 3H, C19-CH₃),1.96 ppm (s, 3H, C18-CH₃), 2.18 ppm (d, 2H, C14-CH₂), 2.43 ppm (s, 3H,C22-CH₃), 2.64 (t, 4H, NHS—CH₂CH₂), 2.92 (s, 2H, aconitic-CH₂), 4.21 ppm(d, 1H, C20-CH_(a)), 4.24 ppm (m, 1H, C7-CH), 4.32 ppm (d, 1H,C20-CH_(b)), 4.95 ppm (dd, 1H, C5CH), 5.23 ppm (d, 1H, C10-CH), 5.40 ppm(d, 1H, C30-CH), 5.69 ppm (d, 1H, C2-CH), 6.40-6.68 (m, 1H,aconitic-CH), 7.51 ppm (m, 2H, C33, C27-CH), 7.53 (m, 6H, C32, C34-CH;C31, C35-CH; C26, C28-CH), 8.12 (d, 2H, C25, C29-CH).

EXAMPLE 11 Synthesis of 2′-aconitic paclitaxel-NHS ester

According to the same procedure as described in Example 10, the desiredproduct of the precursor composed of 2′-acconitic-paclitaxel-NHS esterwas prepared using aconitic anhydride (3.12 g, 20 mmol), paclitaxel(8.53 g, 10 mmol), DPTS (5.88 g, 20 mmol), NHS (11.5 g, 100 mmol), DIC(2.52 g, 20 mmol) and DIPEA (10 ml).

Composition: C₅₅H₅₈N₂O₁₉

Elemental analysis data (%): C, (61.90); H, (5.75); N, (2.80).Theoretical value: (62.85); H, (5.56); N, (2.67).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.13 ppm (s, 3H, C17-CH₃), 1.24 ppm (s,3H, C16-CH₃), 1.34 ppm (s, 9H, C60-t Bu), 1.75 ppm (s, 3H, C19-CH₃),1.96 ppm (s, 3H, C18-CH₃), 2.18 ppm (d, 2H, C14-CH₂), 2.43 ppm (s, 3H,C22-CH₃), 2.64 (t, 4H, NHS—CH₂CH₂), 2.92 (s, 2H, aconitic-CH₂), 4.21 ppm(d, 1H, C20-CH_(a)), 4.24 ppm (m, 1H, C7-CH), 4.32 ppm (d, 1H,C20-CH_(b)), 4.95 ppm (dd, 1H, C5-CH), 5.23 ppm (d, 1H, C10-CH), 5.40ppm (d, 1H, C30-CH), 5.69 ppm (d, 1H, C2-CH), 6.40-6.68 (m, 1H,aconitic-CH), 7.51 ppm (m, 2H, C33, C27-CH), 7.53 (m, 6H, C32, C34-CH;C31, C35-CH; C26, C28-CH), 8.12 (d, 2H, C25, C29-CH).

EXAMPLE 12 Synthesis of 2′-aconitic camptothecin-NHS ester

According to the same procedure as described in Example 10, the desiredproduct of the precursor composed of 2′-acconitic-camptothecin-NHS esterwas prepared using aconitic anhydride (3.12 g, 20 mmol), camptothecin(3.48 g, 10 mmol), DPTS (5.88 g, 20 mmol), NHS (11.5 g, 100 mmol), DIC(2.52 g, 20 mmol) and DIPEA (10 ml).

Composition: C₃₀H₂₃N₃O₁₁

Elemental analysis data (%): C, (60.29); H, (3.98); N, (6.57).Theoretical value: (59.90); H, (3.85); N, (6.99).

¹H-NMR spectra (CDCl₃) (δ, ppm): 0.9 (t, 3H, C18-CH₃), 2.0 (m, 2H,C19-CH₂), 2.64 (t, 4H, NHS—CH₂CH₂), 2.92 (s, 2H, aconitic-CH₂), 4.20 (d,2H, C5-CH₂), 4.76 (m, 2H, C22-CH₂), 6.40-6.68 (m, 1H, aconitic-CH), 6.70(s, 1H, C14-CH), 7.59 (s, 1H, C11-CH), 7.80 (m, 2H, C12-CH; C7-CH), 8.0(m, 2H, C9-CH; C12-CH).

EXAMPLE 13 Synthesis of 2′-aconitic glycamptothecin

t-Boc-glycine (0.5 g, 2.85 mmol) and camptothecin(CPT) (0.5 g, 1.43mmol) were dissolved in anhydrous methylenechloride (20 ml) and theretoDIPC (0.36 ml, 2.85 mmol) and DMAP (0.31 g, 2.53 mmol) were added. Themixture solution was reacted by stirring at room temperature for 16 h.The resulting mixture was extracted by shaking with dilute aqueoushydrochloric acid solution (pH=2). The aqueous layer was separated anddried using anhydrous magnesiun sulfate, followed by vacuum drying toobtain t-Boc-glycamptothecin. This intermediate was reacted in themixture of methylenechloride and trifluoroacetic acid (10 ml/10 ml) for1 h to remove t-Boc group, and the resultant camptothecin-gly-NH₂ andcis-aconitic anhydride (0.57 g, 3.63 mmol) are reacted indimethylformamide solvent (2 ml) at 0° C. for 16 h. To the reactionmixture excess amount of ethyl ether was added to precipitate thecamptothecin precursor, which was filtered and vacuum-dried to obtain2′-aconitic-glycamptothecin in 80% yield.

Composition: C₂₈H₂₃N₃O₁₀

Elemental analysis data (%): C, (59.72); H, (4.01); N, (7.39).Theoretical value: (59.84); H, (4.09); N, (7.48).

¹H-NMR spectra (CDCl₃) (δ, ppm): 0.88-0.93 (brm, 3H, —CH₃ of CPT-C18),2.12-2.16 (m, 2H, —CH₂ of CPT-C-19), 2.86 (s, 2H, —CH₂ ofcis-aconitate), 3.92-4.42 (brm, 2H, —CH₂ of glycine), 5 0.27 (brs, 2H,—CH₂ of CPT-C5), 5.49 (brs, 2H, —CH₂ of CPT-C22), 5.97 (s, 1H, ═CH ofcis-aconitate), 7.18-7.21 (m, 1H, ═CH of CPT-C14), 7.69-7.72 (m, 1H, ═CHof CPT-C11), 7.84-7.89 (m, 1H, ═CH of CPT-C10) 8.10-8.21 (m, 2H, ═CH ofCPT-C12 and C9), 8.68 (brs, 1H, ═CH of CPT-C7), 12.3-12.8 (brs, —COOH ofcis-aconitatic acid).

Synthesis of Polyphosphazene-Anticancer Drug Conjugates

EXAMPLE 14 Synthesis of[NP(MPEG550)_(1.5)(LysEt-2′-aconitic-docetaxel)_(0.5)]_(n)

The polyphosphazene compound (4.85 g. 5.0 mmol) obtained in Example 1was dissolved in methylene chloride in a reaction flask, which wascooled using ice bath. The 2′-aconitic-docetaxel-NHS ester obtained inExample 8 dissolved also in methylene chloride was added to the reactionflask and the reaction mixture was reacted for 12 h at low temperature(0-5° C.). After 12 h reaction, the reaction mixture was subjected tovacuum evaporation and the residue was dissolved in a small amount ofethanol, followed by addition of a large excess amount of water forrecrystallization, which was repeated twice. The remaining insolubleimpurities were removed using membrane filter and the filtrate waswashed five times with 30% ethanol aqueous solution and then five timeswith pure water, followed by fractionation using ultra-membrane andfreeze-dry to obtain the final polyphosphazene-docetaxel conjugate,[NP(MPEG550)_(1.5)(LysEt-2′-aconitic-docetaxel)_(0.5)]_(n) in 90% yield.

Composition: C₁₃₂H₂₂₅N₅O₅₉P₂.

Elemental analysis data (%): C, (53.62); H, (7.92); N, (2.67).Theoretical value: (54.89); H, (7.85); N, (2.42).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.13 ppm (s, 1.5H, C17-CH₃), 1.24 ppm(s, 1.5H, C16-CH₃), 1.34 ppm (bs, 4.1H, C60-t Bu), 1.75 ppm (s, 1.5H,C19-CH₃), 1.96 ppm (s, 1.5H, C18-CH₃), 2.18 ppm (d, 1.0H, C14-CH₂), 2.43ppm (s, 1.5H, C22-CH₃), 4.21 ppm (d, 0.5H, C20-CH_(a)), 4.24 ppm (m,0.5H, C7-CH), 4.32 ppm (d, 0.5H, C20-CH_(b)), 4.95 ppm (dd, 0.5H,C5-CH), 5.23 ppm (d, 0.5H, C10-CH), 5.40 ppm (d, 0.5H, C30-CH), 5.69 ppm(d, 0.5H, C2-CH), 7.51 ppm (m, 1.0H, C33, C27-CH), 7.53 (m, 3.0H, C32,C34-CH; C31, C35-CH; C26, C28-CH), 8.12 (d, 0.6H, C25, C29-CH), 1.24 (s,1.5H, Lys-OCH₂CH₃), 1.29 (bs, 1H, Lys-CH₂), 1.55 ppm (bs, 1H, Lys-CH₂),1.80 (bs, 1H, Lys-CH₂), 2.90 (br, 1H, Lys-e-CH₂), 3.38 ppm (s, 4.50H,CH₃O—, PEG), and 3.63 ppm (m, 66.0H, —CH₂CH₂—O—), 4.4 (s, 0.51H,Lysine-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 15 Synthesis of[NP(MPEG550)_(1.5)(LysEt)_(0.2)(LysEt-2′-aconitic-docetaxel)_(0.3)]_(n)

According to the same procedure as described in Example 14, the desiredtitle product was prepared using the polyphosphazene compound of Example1 (9.7 g, 10 mmol), the precursor, 2′-aconitic docetaxel NHS ester (3.18g, 3.0 mmol) of Example 11 and DIPEA (5 ml) in 90% yield.

Composition: C_(112.4)H₂₀₃N_(4.6)O_(51.8)P₂.

Elemental analysis data (%): C, (53.48); H, (8.31); N, (2.64).Theoretical value: (53.79); H, (8.15); N, (2.57).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.13 ppm (s, 0.9H, C17-CH₃), 1.24 ppm(s, 0.9H, C16-CH₃), 1.34 ppm (bs, 3.31H, C60-t Bu), 1.75 ppm (s, 0.9H,C19-CH₃), 1.96 ppm (s, 0.9H, C18-CH₃), 2.18 ppm (d, 0.6H, C14-CH₂), 2.43ppm (s, 0.9H, C22-CH₃), 4.21 ppm (d, 0.3H, C20-CH_(a)), 4.24 ppm (m,0.3H, C7-CH), 4.32 ppm (d, 0.3H, C20-CH_(b)), 4.95 ppm (dd, 0.31H,C5-CH), 5.23 ppm (d, 0.3H, C10-CH), 5.40 ppm (d, 0.3H, C30-CH), 5.69 ppm(d, 0.3H, C2-CH), 7.51 ppm (m, 0.6H, C33, C27-CH), 7.53 (m, 1.8H, C32,C34-CH; C31, C35-CH; C26, C28-CH), 8.12 (d, 0.6H, C25, C29-CH), 1.24 (s,1.5H, Lys-OCH₂CH₃), 1.29 (bs, 1H, Lys-CH₂), 1.55 ppm (bs, 1H, Lys-CH₂),1.80 (bs, 1H, Lys-CH₂), 2.90 (br, 1H, Lys-e-CH₂), 3.38 ppm (s, 4.50H,CH₃O—, PEG), and 3.63 ppm (m, 66.0H, —CH₂CH₂—O—), 4.4 (s, 0.51H,Lysine-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 16[NP(MPEG750)_(1.5)(LysEt)_(0.2)(LysEt-2′-aconitic-docetaxel)_(0.3)]_(n)

According to the same procedure as described in Example 14, the desiredtitle product was prepared using the polyphosphazene compound of Example2 (12.3 g, 10 mmol), the precursor, 2′-aconitic docetaxel NHS ester (5.3g, 5.0 mmol) of Example 11 and DIPEA (10 ml) in 89% yield.

Composition: C_(136.4)H₂₅₁N_(4.6)O_(63.8)P₂.

Elemental analysis data (%): C, (53.27); H, (8.45); N, (2.31).Theoretical value: (53.92); H, (8.33); N, (2.12).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.13 ppm (s, 0.9H, C17-CH₃), 1.24 ppm(s, 0.9H, C16-CH₃), 1.34 ppm (bs, 3.31H, C60-t Bu), 1.75 ppm (s, 0.9H,C19-CH₃), 1.96 ppm (s, 0.9H, C18-CH₃), 2.18 ppm (d, 0.6H, C14-CH₂), 2.43ppm (s, 0.9H, C22-CH₃), 4.21 ppm (d, 0.3H, C20-CH_(a)), 4.24 ppm (m,0.3H, C7-CH), 4.32 ppm (d, 0.3H, C20-CH_(b)), 4.95 ppm (dd, 0.31H,C5-CH), 5.23 ppm (d, 0.3H, C10-CH), 5.40 ppm (d, 0.3H, C30-CH), 5.69 ppm(d, 0.3H, C2-CH), 7.51 ppm (m, 0.6H, C33, C27-CH), 7.53 (m, 1.8H, C32,C34-CH; C31, C35-CH; C26, C28-CH), 8.12 (d, 0.6H, C25, C29-CH), 1.24 (s,1.5H, Lys-OCH₂CH₃), 1.29 (bs, 1H, Lys-CH₂), 1.55 ppm (bs, 1H, Lys-CH₂),1.80 (bs, 1H, Lys-CH₂), 2.90 (br, 1H, Lys-ε-CH₂), 3.38 ppm (s, 4.50H,CH₃O—, PEG), and 3.63 ppm (m, 98.0H, —CH₂CH₂O—), 4.0 (bs, 4H, MPEG750-CH₂), 4.51 (s, 0.51H, Lysine-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 17[NP(MPEG1000)_(1.5)(LysEt)_(0.2)(LysEt-2′-aconitic-docetaxel)_(0.3)]_(n)

According to the same procedure as described in Example 14, the desiredtitle product was prepared using the polyphosphazene compound of Example3 (15.6 g, 10 mmol), the precursor, 2′-aconitic docetaxel NHS ester (5.3g, 5.0 mmol) of Example 11 and DIPEA (10 ml) in 89% yield.

Composition: C_(172.4)H₃₂₃N_(4.6)O_(48.2)P₂.

Elemental analysis data (%): C, (53.71); H, (8.74); N, (2.01).Theoretical value: (54.04); H, (8.50); N, (1.68).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.13 ppm (s, 0.9H, C17-CH₃), 1.24 ppm(s, 0.9H, C16-CH₃), 1.34 ppm (bs, 3.31H, C60-t Bu), 1.75 ppm (s, 0.9H,C19-CH₃), 1.96 ppm (s, 0.9H, C18-CH₃), 2.18 ppm (d, 0.6H, C14-CH₂), 2.43ppm (s, 0.9H, C22-CH₃), 4.21 ppm (d, 0.3H, C20-CH_(a)), 4.24 ppm (m,0.3H, C7-CH), 4.32 ppm (d, 0.3H, C20-CH_(b)), 4.95 ppm (dd, 0.31H,C5-CH), 5.23 ppm (d, 0.3H, C10-CH), 5.40 ppm (d, 0.3H, C30-CH), 5.69 ppm(d, 0.3H, C2-CH), 7.51 ppm (m, 0.6H, C33, C27-CH), 7.53 (m, 1.8H, C32,C34-CH; C31, C35-CH; C26, C28-CH), 8.12 (d, 0.6H, C25, C29-CH), 1.24 (s,1.5H, Lys-OCH₂CH₃), 1.29 (bs, 1H, Lys-CH₂), 1.55 ppm (bs, 1H, Lys-CH₂),1.80 (bs, 1H, Lys-CH₂), 2.90 (br, 1H, Lys-ε-CH₂), 3.38 ppm (s, 4.50H,MPEG-CH₃O—), and 3.63 ppm (m, 128H, MPEG-CH₂CH₂O), 4.0 (bs, 4H,MPEG1000-CH₂), 4.51 (s, 0.51H, Lysine-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 18 Synthesis of[NP(MPEG550)_(1.0)(LysEt)_(0.5)(LysEt-2′-aconitic-docetaxel)_(0.5)]_(n)

According to the same procedure as described in Example 14, the desiredtitle product was prepared using the polyphosphazene compound of Example5 (7.7 g, 10 mmol), the precursor, 2′-aconitic docetaxel NHS ester (6.36g, 6.0 mmol) of Example 10 and DIPEA (10 ml) in 89% yield.

Composition: C₁₁₅H₁₉₁N₇O₄₈P₂.

Elemental analysis data (%): C, (54.92); H, (8.01); N, (3.99).Theoretical value: (55.21); H, (7.70); N, (3.92).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.13 ppm (s, 0.9H, C17-CH₃), 1.24 ppm(s, 0.9H, C16-CH₃), 1.34 ppm (bs, 3.31H, C60-t Bu), 1.75 ppm (s, 0.9H,C19-CH₃), 1.96 ppm (s, 0.9H, C18-CH₃), 2.18 ppm (d, 0.6H, C14-CH₂), 2.43ppm (s, 0.9H, C22-CH₃), 4.21 ppm (d, 0.3H, C20-CH_(a)), 4.24 ppm (m,0.3H, C7-CH), 4.32 ppm (d, 0.3H, C20-CH_(b)), 4.95 ppm (dd, 0.31H,C5-CH), 5.23 ppm (d, 0.3H, C10-CH), 5.40 ppm (d, 0.3H, C30-CH), 5.69 ppm(d, 0.3H, C2-CH), 7.51 ppm (m, 0.6H, C33, C27-CH), 7.53 (m, 1.8H, C32,C34-CH; C31, C35-CH; C26, C28-CH), 8.12 (d, 0.6H, C25, C29-CH), 1.24 (s,3.0H, Lys-OCH₂CH₃), 1.29 (bs, 2H, Lys-CH₂), 1.55 ppm (bs, 2H, Lys-CH₂),1.80 (bs, 2H, Lys-CH₂), 2.90 (br, 2H, Lys-e-CH₂), 3.38 ppm (s, 3.00H,CH₃O—, PEG), and 3.63 ppm (m, 44.0H, —CH₂CH₂—O—), 4.4 (s, 0.51H,Lysine-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 19 Synthesis of[NP(MPEG550)_(1.5)(GlyLysEt)}_(0.2)(GlyLysEt-2′-aconitic-ocetaxel)_(0.3)]_(n)

According to the same procedure as described in Example 14, the desiredtitle product was prepared using the polyphosphazene compound of Example6 (10.4 g, 10 mmol), the precursor, 2′-aconitic docetaxel NHS ester (5.3g, 5.0 mmol) of Example 10 and DIPEA (10 ml) in 89% yield.

Composition: C_(114.4)H₂₀₆N_(5.6)O_(52.8)P₂.

Elemental analysis data (%): C, (52.98); H, (8.23); N, (3.19).Theoretical value: (53.53); H, (8.09); N, (3.06).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.13 ppm (s, 0.9H, C17-CH₃), 1.24 ppm(s, 0.9H, C16-CH₃), 1.34 ppm (bs, 3.31H, C60-t Bu), 1.75 ppm (s, 0.9H,C19-CH₃), 1.96 ppm (s, 0.9H, C18-CH₃), 2.18 ppm (d, 0.6H, C14-CH₂), 2.43ppm (s, 0.9H, C22-CH₃), 4.21 ppm (d, 0.3H, C20-CH_(a)), 4.24 ppm (m,0.3H, C7-CH), 4.32 ppm (d, 0.3H, C20-CH_(b)), 4.95 ppm (dd, 0.31H,C5-CH), 5.23 ppm (d, 0.3H, C10-CH), 5.40 ppm (d, 0.3H, C30-CH), 5.69 ppm(d, 0.3H, C2-CH), 7.51 ppm (m, 0.6H, C33, C27-CH), 7.53 (m, 1.8H, C32,C34-CH; C31, C35-CH; C26, C28-CH), 8.12 (d, 0.6H, C25, C29-CH), 1.24 (s,1.5H, Lys-OCH₂CH₃), 1.29 (bs, 1H, Lys-CH₂), 1.55 ppm (bs, 1H, Lys-CH₂),1.80 (bs, 1H, Lys-CH₂), 2.90 (br, 1H, Lys-e-CH₂), 3.38 ppm (s, 4.50H,CH₃O—, PEG), and 3.63 ppm (m, 66.0H, —CH₂CH₂—O—), 3.98 (bs, 2H,Gly-CH₂), 4.4 (s, 0.51H, Lysine-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 20 Synthesis of[NP(MPEG550)_(1.50)(LysEt)_(0.2)(LysEt-2′-succinylpaclitaxel)_(0.3)]_(n)

The polyphosphazene compound of Example 1 (9.7 g, 10 mmol) was reactedwith 2′-succinylpaclitaxel (5.26 g, 5.0 mmol) prepared by the literatureprocedure (C.-M. Huang, et al, Chem. Biol. 2000, 7, 453-461) byesterification using DCL (2.54 g, 20 mmol) and DIPEA (10 ml) to obtainthe desired title compound in 90% yield.

Composition: C_(113.6)H₂₀₂N_(4.6)O_(51.8)P₂.

Elemental analysis data (%): C, (54.15); H, (8.26); N, (2.61).Theoretical value: (54.49); H, (8.12); N, (2.57).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.25 (s, 1.5H, Lys-OCH₂CH₃), 2.49 (br,1.00H, suucinyl-CH₂), 2.90 (br, 1.00H, Lys-ε-CH₂), 3.38 (s, 4.50H,MPEG550-OCH₃), 3.65 (br, 66.0H, MPEG550-OCH₂CH₂), 1.13 (s, 12H), 1.25(s, 12H), 1.35 (s, 36H), 1.68 (m, 8H), 1.75 (s, 12H), 1.86 (m, 8H), 1.96(s, 12H), 2.36 (m, 20H), 2.60 (m, 4H), 3.98 (s, 8H), 4.06 (d, 8H), 4.30(m, 12H), 4.33 (m. 8H), 4.97 (d, 4H), 5.22 (m, 4H), 5.36 (s, 4H), 5.60(m, 4H), 5.69 (m, 8H), 6.20 (t, 4H), 7.33 (m, 8H), 7.41 (m, 8H), 7.52(m, 8H), 7.61 (m, 4H), 7.33 (m, 4H), 8.12 (d, 8H).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 21 Synthesis of[NP(MPEG550)_(1.50)(GlyLysEt-2′-succinylpaclitaxel)_(0.50)]_(n)

According to the same procedure as described in Example 20, the desiredtitle product was prepared using the polyphosphazene compound of Example6 (10.4 g, 10 mmol), 2′-succinylpaclitaxel (7.36 g, 7.0 mmol) preparedby the literature procedure (C.-M. Huang, et al, Chem. Biol. 2000, 7,453-461) by esterification reaction using DCL (2.54 g, 20 mmol) andDIPEA (10 ml) to obtain the desired title compound in 80% yield.

Composition: C₁₃₆H₂₂₆N₆O₅₈P₂.

Elemental analysis data (%): C, (55.36); H, (7.99); N, (2.93).Theoretical value: (55.67); H, (7.76); N, (2.86).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.25 (s, 1.5H, Lys-OCH₂CH₃), 2.49 (br,1.00H, suucinyl-CH₂), 2.90 (br, 1.00H, Lys-ε-CH₂), 3.38 (s, 4.50H,MPEG550-OCH₃), 3.65 (br, 66.0H, MPEG550-OCH₂CH₂), 1.13 (s, 12H), 1.25(s, 12H), 1.35 (s, 36H), 1.68 (m, 8H), 1.75 (s, 12H), 1.86 (m, 8H), 1.96(s, 12H), 2.36 (m, 20H), 2.60 (m, 4H), 3.98 (s, 8H), 4.06 (d, 8H), 4.30(m, 12H), 4.33 (m. 8H), 4.97 (d, 4H), 5.22 (m, 4H), 5.36 (s, 4H), 5.60(m, 4H), 5.69 (m, 8H), 6.20 (t, 4H), 7.33 (m, 8H), 7.41 (m, 38H), 7.52(m, 8H), 7.61 (m, 4H), 7.33 (m, 4H), 8.12 (d, 8H)

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 22 Synthesis of[NP(MPEG550)_(1.50)(N_(α)-BocLys)_(0.2)(N_(α)-BocLys-docetaxel)₀₃]_(n)

The polyphosphazene compound of Example 7 (9.57 g. 10.0 mmol) anddocetaxel (10.6 g, 10.0 mmol) are vacuum dried and then dissolved in adried solvent of tetrahydrofuran, methylene chloride or chloroform in areaction vessel, which was cooled in ice bath and then the catalyst DC1(2.54 g, 20 mmol) and DIPEA (10 ml) dissolved in the same solvent wereadded thereto. After the reaction mixture was reacted at ice temperaturefor 24 h, the reaction solution was filtered at reduced pressure and thefiltrate was vacuum dried. The resultant product was purified in thesame way as in Example 14 to obtain the title compound in 60% yield.

Composition: C_(113.8)H_(204.2)N_(4.6)O_(50.8)P₂.

Elemental analysis data (%): C, (55.06); H, (7.98); N, (2.60).Theoretical value: (54.42); H, (8.19); N, (2.57).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.25 (s, 1.5H, Lys-OCH₂CH₃), 2.49 (br,1.00H, suucinyl-CH₂), 2.90 (br, 1.00H, Lys-ε-CH₂), 3.38 (s, 4.50H,MPEG550-OCH₃), 3.65 (br, 66.0H, MPEG550-OCH₂CH₂), 1.13 (s, 12H), 1.25(s, 12H), 1.35 (s, 36H), 1.68 (m, 8H), 1.75 (s, 12H), 1.86 (m, 8H), 1.96(s, 12H), 2.36 (m, 20H), 2.60 (m, 4H), 3.98 (s, 8H), 4.06 (d, 8H), 4.30(m, 12H), 4.33 (m. 8H), 4.97 (d, 4H), 5.22 (m, 4H), 5.36 (s, 4H), 5.60(m, 4H), 5.69 (m, 8H), 6.20 (t, 4H), 7.33 (m, 8H), 7.41 (m, 8H), 7.52(m, 8H), 7.61 (m, 4H), 7.33 (m, 4H), 8.12 (d, 8H).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 23 Synthesis of[NP(MPEG550)_(1.5)(LysEt)_(0.2)(LysEt-2′-aconitic-camptothecin)_(0.3)]_(n)

The polyphosphazene compound of Example 1 (9.7 g, 10 mmol) and2′-aconitic-amptothecin-NHS ester (2.5. g, 5.03 mmol) of Example 12 werereacted according to the same method of Example 14 the required titlecompound was obtained in 75% yield.

Composition: C_(98.6)H_(180.8)N_(5.2)O_(45.8)P₂.

Elemental analysis data (%): C, (52.61); H, (8.42); N, (3.34).Theoretical value: (53.01); H, (8.16); N, (3.26).

¹H-NMR spectra (CDCl₃) (δ, ppm): 0.9 (t, 3H, C18-CH3), 2.0 (m, 2H,C19-CH₂), 2.64 (t, 4H, NHS—CH₂CH₂), 2.92 (s, 2H, aconitic-CH₂), 4.20 (d,2H, C5-CH₂), 4.76 (m, 2H, C22-CH₂), 6.40-6.68 (m, 1H, aconitic-CH), 6.70(s, 1H, C14-CH), 7.59 (s, 1H, C11-CH), 7.80 (m, 2H, C12-CH; C7-CH), 8.0(m, 2H, C9-CH; C12-CH), 1.24 (s, 1.5H, Lys-OCH₂CH₃), 1.29 (bs, 1H,Lys-CH₂), 1.55 ppm (bs, 1H, Lys-CH₂), 1.80 (bs, 39 1H, Lys-CH₂), 2.90(br, 1H, Lys-e-CH₂), 3.38 ppm (s, 4.50H, CH₃O—, PEG), and 3.63 ppm (m,66.0H, —CH₂CH₂—O—), 4.4 (s, 0.51H, Lysine-CH).

³¹P-NMR spectra: (CDCl₃, ppm): δ −0.014 (s), δ -5.551 (s).

EXAMPLE 24 Synthesis of[NP(MPEG550)(LysEt)(aconitic-glycylcamptothecin)]_(n)

According to the same procedure as described in Example 14, the desiredtitle product was prepared using the polyphosphazene compound of Example5 (0.5 g, 0.25 mmol) and the precursor, 2′-aconitic-glycamptothecin(0.17 g, 0.25 mmol) of Example 13 and DIPEA (10 ml) in 85% yield.

Composition: C₁₁₁H₁₉₁N₇O₅₀P₂.

¹H-NMR spectra (CDCl₃) (δ, ppm): 0.89-0.92 (brm, 3H, —CH₃ of CPT-C18),1.13-1.58 (brm, 6H, —CH₂ of lysine), 2.12-2.17 (brm, 2H, —CH₂ ofCPT-C-19), 3.01 (s, 2H, —CH₂ of cis-aconitate), 3.21 (s, 9H, —OCH₃ ofMPEG), 3.34-3.54 (brm, 144H, —CH₂-CH₂ of MPEG), 3.94-4.41 (brm, 5H, —CH₂of glycine, P—NH—CH₂ of lysine and ═CH of cis-aconitate), 5.29 (brs, 2H,—CH₂ of CPT-C5), 5.47 (brs, 2H, —CH₂ of CPT-C22), 7.15-7.17 (m, 1H, ═CHof CPT-C14), 7.69-7.72 (m, 1H, ═CH of CPT-C11), 7.84-7.94 (m, 1H, ═CH ofCPT-C10) 8.10-8.21 (m, 2H, ═CH of CPT-C12 and CPT-C9), 8.68 (brs, 1H,═CH of CPT-C7).

³¹P-NMR (DMSO, ppm): δ −5.19 (O—P—O), 0.85 (O—P—N).

EXAMPLE 25 Synthesis of [NP(MPEG550)(AE)(ACA)Pt(dach)]_(n)

The polyphosphazene carrier polymer, [NP(MPEG550)(AE)]_(n) (1 g, 1.5mmol) of Example 8 was dissolved in an aqueous sodium bicarbonatesolution (pH=9.0) and the linker cis-aconitic anhydride (ACA) (2.35 g,15 mmol) was added thereto for further reaction at 4° C. for 5 h. Thereaction mixture was dialyzed using a cellulose membrane (MWCO: 3.5 kDa) to obtain a new polyphosphazene intermediate bearing the linkergroup, [NP(MPEG550)(AE)(ACA)]_(n). A methanol solution (20 ml) of bariumhydroxide (1.33 mmol) was added to the dialyzed solution and thereaction mixture was further stirred for 5 h to convert the acidiclinker group to barium salt of the polymer. The reaction solution wassubjected to vacuum evaporation to dryness. The solid polymer wasdissolved in distilled water (10 ml) and to this polymer solution wasslowly added an aqueous solution (10 ml) of (dach)Pt(SO₄)(dach:trans±1,2-diaminocyclohexane) (0.49 g, 1.21 mmol) and the reactionmixture was further stirred for 3 h. After the barium sulfateprecipitate was filtered out, the filtrate was dialyzed using cellulosemembrane (MWCO: 3.5 kDa) and subjected to freeze dry to obtain a novelpolyphosphazene-oxaliplatin conjugate drug in 74% yield.

Composition: C₃₉H₇₃N₄O₁₉PPt.H₂O

Elemental analysis data (%): C, (40.54); H, (6.34); N, (4.62).Theoretical value: (40.83); H, (6.54); N, (4.88).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.04-1.20 (brm, 4H, C-4, C-5 of dach),1.44 (brs, 2H, C-3 of dach), 1.82-1.93 (brm, 2H, C-6 of dach), 2.02-2.52(brm, 2H, C-1, C-2 of dach), 3.26 (s, 3H, OCH₃ of MPEG), 3.41-3.45 (m,6H, CH₂ of cis-aconitate and aminoethanol), 3.47-3.81 (brm, 46H, —O—CH₂of MPEG), 3.95-4.21 (brm, 2H, —P—O—CH₂— of MPEG), 4.69 (s, 1H, —C═CH— ofcis-aconitate).

³¹P-NMR (DMSO, ppm): −4.53 (O—P—O).

EXAMPLE 26 Synthesis of [NP(MPEG750)(AE)(ACA)Pt(dach)]_(n)

According to the same procedure as described in Example 25, the desiredtitle product was prepared using the polyphosphazene compound of Example9 (1.0 g, 11.91 mmol), cis-aconitic anhydride (1.86 g, 1.191 mmol),Ba(OH)₂.8H₂O (0.35 g, 1.11 mmol), and (dach)PtSO₄ (0.4 g, 0.99 mmol) in72% yield.

Composition: C₄₇H₈₉N₄O₂₃PPt.H₂O

Elemental analysis data (%): C, (42.32); H, (7.64); N, (3.88).Theoretical value: (42.65); H, (6.88); N, (4.23).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.05-1.21 (brm, 4H, C-4, C-5 of dach),1.47 (brs, 2H, C-3 of dach), 1.80-1.93 (brm, 2H, C-6 of dach), 2.01-2.45(brm, 2H, C-1, C-2 of dach), 3.27 (s, 3H, —OCH₃ of MPEG), 3.41-3.45 (m,6H, —CH₂ of cis-aconitate and aminoethanol), 3.50-3.72 (brm, 62H, —CH₂of MPEG), 3.99-4.13 (brm, 2H, —P—O—CH₂ of MPEG), 4.70 (s, ₁H, —C═CH— ofcis-aconitate).

³¹P-NMR (DMSO, ppm): −4.52 (O—P—O).

EXAMPLE 27 Synthesis of [NP(MPEG550)(LysEt)(ACA)Pt(dach)]_(n)

According to the same procedure as described in Example 25, the desiredtitle product was prepared using the polyphosphazene compound of Example5 (1.0 g, 1.28 mmol), cis-aconitic anhydride (2 g, 1.191 mmol),Ba(OH)₂.8H₂O (0.44 g, 1.39 mmol), and (dach)PtSO₄ (0.52 g, 1.28 mmol) in79% yield.

Composition: C₄₅H₈₄N₅O₂₀PPt.H₂O

Elemental analysis data (%): C, (42.63); H, (6.61); N, (5.42).Theoretical value: (42.88); H, (6.83); N, (5.56).

¹H-NMR spectra (CDCl₃) (δ, ppm): 1.07-1.21 (brm, 7H, —CH₃, —(CH₂)₂ oflysine), 1.43-1.48 (brm, 6H, —C-3, —C-4, C-5 of dach), 1.80-1.93 (brm,8H, —CH₂ lysine and —C-6 of dach), 2.02-2.26 (brm, 2H, C-1, C-2 ofdach), 2.82 (brs, 2H, —CH₂ of cis-aconitate), 3.26 (s, 3H, —OCH₃ ofMPEG), 3.44-3.72 (brm, 46H, —CH₂—CH₂ of MPEG), 3.96-4.03 (brm, 3H, —CH₂of ethylester and —N—CH of lysine), 4.62 (s, 1H, ═CH of cis-aconitate).

Measurements of Physicochemical Properties and Drug Efficacy

EXPERIMENTAL 1 Particle Size and Micelle Formation

The polyphosphazene carrier compound of Example 1 and thepolyphosphazene-docetaxel conjugate of Example 14 were separatelydissolved in distilled water (0.2%) and their particle sizedistributions and zeta potentials were measured by DLS (dynamic lightscattering) method and the results are displayed in FIGS. 1, 2, and 3.

FIG. 1 shows the particle size distribution of the polyphosphazenecarrier polymer with a mean diameter of approximately 3˜4 nm, whichcorresponds to the particle size of a hydrodynamic volume of unassembledpolymers probably due to the cationic properties of the lysine aminegroup of the polymer as shown in FIG. 2. However, the increased particlesize of the polyphosphazene-docetaxel conjugate to 60 nm as shown inFIG. 3 clearly indicates that the conjugate drug molecules wereself-assembled into larger micellar nanoparticles attributed to theamphiphilic properties of the conjugate molecules by introduction ofhydrophobic docetaxel molecules into the carrier polymer. It was furtherconfirmed that the micelle size was not changed significantly in thetemperature range of 5˜70° C.

EXPERIMENTAL 2 Measurement of the Critical Micelle Concentration (CMC)of Polyphosphazene-Paclitaxel Conjugate

As above-mentioned, the polyphosphazene-docetaxel conjugate of thepresent invention forms polymeric micelles in aqueous solution. In orderfor such polymer-drug conjugate to be clinically useful for IVinjection, the solution stability of the micelles self-assembled fromthe polymer-drug conjugate is critical. Such a micelle stability isexpressed as “critical micelle concentration (CMC),” which is measuredby several methods, but the most widely used method is “pyrenefluorescence method” (K. Kalyanasundoram, et. al, J. Am. Chem. Soc.1988, 99, 2039). According to the method the CMC value of thepolyphosphazene-docetaxel conjugate of the present invention wasmeasured as in the following:

An aqueous pyrene solution (6×10⁻⁷ M) was prepared and using thissolution a series of sample solutions of the polyphosphazene-paclitaxelconjugate of Example 20 was prepared in the concentration range of5.0˜0.0005% (w/w). Fluorescence spectra were measured at 339 nm (I_(ex))and 390 nm (I_(em)) and then from the ratio of the fluorescenceintensities of band I and band III, the value of CMC was determined asshown in FIG. 4.

The CMC value of the polyphosphazene-paclitaxel conjugate thus obtainedwas 41 mg/L, which is very low and the polymeric micelles are expectedto be stable in the blood system when injected intravenously.

EXPERIMENTAL 3 Biodegradability of Polyphosphazene-Drug Conjugate

-   Instrument: Yonglin GPC system-   Column: Waters Hydrogel HR column (1× guard, 1× linear, 2×HR2)-   Eluent: water/acetonitrile (8:2) (0.5% NaNO₃)-   Flow rate: 1 ml/min.

Two sample solutions were prepared by dissolving thepolyphosphazene-paclitaxel conjugate of Example 20 (250 mg) in a buffersolution (5 ml) at pH=5.4 and in another buffer solution (5 ml) atpH=7.4. The two sample solutions were slowly shaken at 37° C. in a waterbath and 500 μl of the sample solution was taken at predeterminedschedule (0.5, 1, 2, 4, 6, 8, 16 day after incubation) and freeze-dried.The dried samples were dissolved in tetrahydrofuran involving 0.2%t-butyl ammonium bromide and subjected to gel permeation chromatography(GPC). From the GPC data the degradation pattern ofpolyphosphazene-paclitaxel conjugate drug is shown in FIG. 5.

As seen from the figure, the average molecular weight of thepolyphosphazene conjugate drug rapidly decreased for first a few days,but its degradation was slow down particularly at neutral medium. Thehalf-life of the polyphosphazene backbone in blood system was estimatedto be approximately 16 days but the most encouraging factor is that thepolyphosphazene backbone is continuously degradable in acidic media,which is similar to the tumor microenvironment relevant to the drugreleasing kinetics. In the present invention all the final polymerproducts were subjected to fractionation into different molecularweights to study their biodegradation, excretion, and drug releasingkinetics.

EXPERIMENTAL 4 Tumor Targeting Properties of Polyphosphazene Compound byImaging Study

-   Instrument: Kodak image station 4000 mm digital imaging system    (Kodak, New Haven, Conn.) Excitation and emission filter: Omega    Optical, Battlebor, Vt. (ex: 560 nm, em: 700 nm) Ten eight weeks old    CH₃/HeN nude mice (Institute of Medical Science, Tokyo) were    purchased and after adaptation for a week inoculated with non-small    cell lung carcinoma A549 (1×10₆). When the tumor size was grown up    to about 300 mm³, the mice were classified into two groups, one    group were injected with fluorescence dye Cy5.5 labeled    polyphosphazene carrier compound of Example 1 and another group    untreated was used as reference group. At predetermined time (12 h,    24 h, 48 h, 72 h after IV injection) the mice were sacrificed to    separate the whole major organs (liver, lung, kidney, spleen, tumor,    muscle), which were subjected to NIR fluorescence image study using    CCD camera (Kodak Image Station 4000MM), and the results are    displayed in FIG. 6.

From the fluorescence intensities of the organs in the figure, thepolyphosphazene compound of Example 1 clearly showed dominantaccumulation in the tumor tissue compared with other organs, despite itssmall particle size of 3-4 nm, which cannot afford EPR effect.Therefore, it may be presumed that the tumor selectivity of thispolyphosphazene carrier polymer is due to its cationic propertiesattributed to the lysine free amine of the polymer and its long bloodcirculation due to its PEGylated structure.

EXPERIMENTAL 5 Tumor Targeting Properties of thePolyphosphazene-Docetaxel Conjugate Drug

The polyphosphazene-docetaxel conjugate of Example 14 was labeled withCy5.5 and its organ distributions were compared in the same way as inExperimental 4 (FIG. 7). The quantitative biodistribution data of theconjugate drug were obtained by comparison of the fluorescence intensityof each organ of the mouse treated with Cy5.5-labeled conjugate drugwith that of the mouse untreated, and the results are displayed in FIG.8.

FIG. 7 shows that the conjugate drug accumulated dominantly in tumor andin particular, its accumulation reached a maximum after 48 hpost-injection probably due to EPR (Enhanced Permeability and Retentioneffect) effect of the larger particle size of the conjugate (60 nm) andlong blood circulation. The quantitative data for biodistributions ofthe polyphosphazene-docetaxel conjugate drug shown in FIG. 8 clearlyshows much higher tumor selectivity of the conjugate drug due to the EPReffect of its large particle size compared with the tumor selectivity ofthe former polyphosphazene carrier polymer attributed to its cationicproperties.

EXPERIMENTAL 6 Analysis of Docetaxel Content in thePolyphosphazene-Docetaxel Conjugate

The content of the drug component in the polyphosphazene-drug conjugatemay be determined by ¹H NMR spectroscopy, UV spectroscopy, or HPLC. Inthe proton NMR spectroscopic method, the docetaxel content in theconjugate drug could be estimated from the ratio of integrated area ofthe methoxy protons at 3.4 ppm (3H —CH₃) of the PEG group grafted to thepolymer backbone and that of the C10 protons at 7.33 ppm (C10, 2H) ofdocetaxel conjugated. However, both chemical shifts of the methoxyprotons and docetaxel C10 protons are slightly overlapped with adjacentproton peaks, which hampers the accuracy of the integration ratio. Inthe HPLC method, the total amount of free docetaxel released from theconjugate drug by acidic decomposition could be measured by HPLC, butreproducible results could not be obtained.

On the other hand, reproducible results could be obtained using UVspectroscopic method, since docetaxel molecule shows a strong UVabsorption at 230 nm while the polyphosphazene carrier polymer exhibitsnearly no absorption at around 230 nm. Therefore, from the calibrationcurve measured using a solvent mixture of water and acetonitrile (1:1),in which docetaxel is completely soluble. Calibration curve was preparedby measuring UV absorption at 230 nm using the standard solution of 10.0mg docetaxel/10 ml solvent mixture and their diluted solutions: 1 mg/ml,½ mg/ml, etc.

EXPERIMENTAL 7 In Vitro Drug Releasing from thePolyphosphazene-Docetaxel Conjugate

-   Instrument: Agilent 1100 series with DAD detector (230 nm)-   Column: Agilent Zobax Eclipse Plus C18 column (diameter=4.6 mm;    length=150 mm, particle size=3.5 μm)-   Flow rate: 1. p ml/min-   Eluent composition: A: 0.1% TFA in H₂O; B: Acetonitrile(isocratic    method)

The in vitro drug releasing experiment of the polyphosphazene-docetaxelconjugate was performed using HPLC method. The amount of released drug,docetaxel from the conjugate was determined using the calibration curveprepared in the same way as in the Experimental 6.

EXPERIMENTAL 8 In Vitro Assay of Cytotoxicity ofPolyphosphazene-Paclitaxel Conjugate

Since the anticancer agent paclitaxel is well known to be clinicallyvery efficacious against many different cancers, breast (MCF-7), ovarian(SK-OV3), non-small cell lung (A549) and stomach (SNU638) cancer celllines were selected to test their in vitro cytotoxicity according to theliterature method (SRB method) (Rita Song, et. al, J. Control. Release105 (2005) 142-150).

The results are presented in the following Table 1. As is seen from thetable, the IC₅₀ values of the polyphosphazene-paclitaxel conjugates ofExample 20 and 21 much higher than the free paclitaxel, since thehydrophobic paclitaxel component is very difficult to be released fromthe polymeric micelle core after degradation of thepolyphosphazene-paclitaxel conjugate.

TABLE 1 In vitro cytotoxicity of polyphosphazene-paclitaxel conjugateIC₅₀ (nM) (mean ± SD, n = 3-4) Test cell lines MCF-7 SK-Ov3 A-549SNU-638 Paclitaxel  3.47 ± 0.62 15.32 ± 2.6  6.63 ± 2.84 10.89 ± 0.90Example 20 164.8 ± 86.5 587.5 ± 47.8 170.0 ± 48.3 364.4 ± 201.7 Example21 316.7 ± 141.1  6964 ± 141.7  1543 ± 41.8  5411 ± 46.6

EXPERIMENTAL 9 Pharmacokinetic Study of the Polyphosphazene-DocetaxelConjugate

In order to examine the pharmacokinetic behavior of thepolyphosphazene-docetaxel conjugate of Example 14 compared with theunconjugated but formulated “Taxotere” currently in clinical use, apharmacokinetic study of the conjugate was performed usingSprague-Dawley rats according to the literature method (Jun et al., Int.J. Pharm. 422(2012) 374-380). The time dependent plasma concentrationprofile was displayed in FIG. 9 and the pharmacokinetic parameters werepresented in Table 2.

TABLE 2 Pharmacokinetic parameters of Example 14 and Taxotere asreference. Taxotere Pharmacokinetic (reference) Example 14 parametersMean SD Mean SD C₀ (μg/mL) 8.764 3.221 0.263 0.051 AUC_(last) (μg ·h/mL) 0.651 0.098 1.192 0.380 AUC_(INF) (μg · h/mL) 0.678 0.098 1.4390.531 t_(1/2) (h) 0.651 0.093 6.115 4.041 V_(z) (L) 1.758 0.159 7.2872.231 Cl (L/h) 1.896 0.255 0.984 0.311

EXPERIMENTAL 10 Nude Mouse Xenograft Trials of thePolyphosphazene-Docetaxel Conjugate

In order to evaluate the in vivo efficacy of Example 14 compared withTaxotere as reference, nude mouse xenograft trials against gastriccancer cell line MKN-28 were performed using BALB/C nude mouse accordingto the literature method (Y. J. Jun, et al. Int. J. Pharm. 422 (2012)374-380). Since the optimal dose of Taxotere is known to be 10 mg/kg,one injection dose of Example 14 was determined to be 10 mg/kg and 20mg/kg based on the docetaxel content of the polyphosphazene-docetaxelconjugate, which were administered three times (day 1, 5, 9). Both tumorsize and body weight of each were measured from the beginning of firstinjection to 40 days. FIG. 10 displays the antitumor activity againstthe MKN-28 gastric tumor cells and FIG. 11 shows the body weight changesduring the 40 days. It is seen from FIG. 10 that thepolyphosphazene-docetaxel conjugate exhibits strong antitumor activityequivalent to Taxotere, and the more important result is seen from FIG.11 showing that the average body weight of the mice treated withTaxotere is significantly reduced (>10%) during the drug injectionperiod while no significant body weight changes are observed for theconjugate drug, which means that the present conjugate drug shows lowersystemic toxicity. The results of xenograft trials performed fornon-small cell lung cancer cell line A549 are displayed in FIG. 12showing that the present conjugate drug exhibits even better antitumoractivity than the reference Taxotere.

The invention claimed is:
 1. A linear polyphosphazene-drug conjugatecompound represented by the following chemical formula 3:

wherein, in chemical formula 3: n is independently an integer from 3 to300; MPEG represents methoxy poly(ethylene glycol); L is a cis-aconiticacid anhydride linker, which connects a LysEt spacer group to drugmolecule D, wherein drug molecule D is docetaxel; R is C₂H₅; x and y areindependently in the range of 0 to 0.5; z is larger than 0 and less than1.0; and x+y+z=1.